Modulators of morphogen expression and methods of identifying the same

ABSTRACT

Disclosed are methods and compositions for identifying compounds having an ability to modulate expression of a morphogen, particularly OP-1, OP-1 homologues and closely related proteins, using one or more OP-1-specific, non-coding sequences and a suitable reporter gene. In preferred embodiments, the OP-1-specific non-coding DNA sequence comprises a Pax-responsive OP-1-modulating element.

This application claims the benefit of Provisional application Ser. No.60/047,911, filed May 29, 1997.

FIELD OF THE INVENTION

The invention relates generally to the field of drug screening assays.More particularly, the invention relates to methods and compositions foridentifying molecules that modulate expression of true tissuemorphogenic proteins.

BACKGROUND OF THE INVENTION

A class of proteins recently has been identified, the members of whichare true tissue morphogenic proteins. The members of this class ofproteins are characterized as competent for inducing the developmentalcascade of cellular and molecular events that culminate in the formationof new organ-specific tissue, including any vascular and connectivetissue formation, as required by the naturally occurring tissue.Specifically, the morphogens are competent for inducing all of thefollowing biological functions in a morphogenically permissiveenvironment: (1) stimulating proliferation of progenitor cells; (2)stimulating differentiation of progenitor cells; (3) stimulating theproliferation of differentiated cells and (4) supporting the growth andmaintenance of differentiated cells. For example, the morphogenicproteins can induce the full developmental cascade of bone tissuemorphogenesis, including the migration and proliferation of mesenchymalcells, proliferation and differentiation of chondrocytes, cartilagematrix formation and calcification, vascular invasion, osteoblastproliferation, bone formation, bone remodeling, and hematopoietic bonemarrow differentiation. These proteins also have been shown to inducetrue tissue morphogenesis of non-chondrogenic tissue, including dentin,liver, and nerve tissue.

A particularly useful tissue morphogenic protein is human OP-1(Osteogenic Protein-1), described in U.S. Pat. No. 5,011,691; U.S. Pat.No. 5,266,683 and Ozkaynak et al. (1990) EMBO J. 9: 2085-2093. Specieshomologues identified to date include, but are not limited to, mouseOP-1 (see U.S. Pat. No. 5,266,683) and the Drosophila homologue 60A,described in Wharton et al. (1991) Proc. Nat. Acad. Sci. USA88:9214-9218). Other closely related proteins include OP-2 (Ozkaynak(1992) J. Biol. Chem. 2:25220-25227 and U.S. Pat. No. 5,266,683); BMP5,BMP6 (Celeste et al. (1991) Proc. Natl. Acad. Sci. 8:9843-9847) andVgr-1 (Lyons et al. (1989). These disclosures are incorporated herein byreference.

It previously has been contemplated that these tissue morphogens can beadministered to an animal to regenerate lost or damaged tissue. Certaincomplications, however, presently are encountered during the production,formulation and use in vivo of therapeutic macromolecules, such asmorphogen proteins. For example, such proteins are typically produced byfermentation or culture of suitable host cells. Any biological productproduced from such host cells for use in humans presently must be shownto be essentially free of host cell contaminants, such as secreted orshed proteins, viral particles or degradation products thereof.Providing such assurance can add significantly to the cost and technicaldifficulty of commercial production of biological macromolecules.Furthermore, appropriate formulations must be developed for conferringcommercially reasonable shelf life on the produced macromolecule,without significant loss of biological efficacy. An additionalcomplicating factor arises when circumstances warrant an extended courseof therapeutic treatment with the produced and formulated macromolecule:the treated mammal may develop an immunological response to themacromolecule, and any such response may interfere with effectivenessthereof. In extreme circumstances, treatment must be discontinued.

Alternatively, administering a molecule capable of modulating expressionof the endogenous tissue morphogen is an effective means for providingmorphogen to a site in vivo. For example, DNA sequences have beenidentified in the OP-1 gene promoter that resemble wt-1/Egr-1 consensussequences, TLC binding sequences, FTZ binding sequences and steroidbinding sequences (see WO 95/33831, the disclosure of which isincorporated herein by reference). Thus, molecules to which theseregulatory sequences are responsive are likely modulators of the OP-1gene and can influence its expression.

It is an object of this invention to provide compositions and methods ofidentifying compounds which can modulate expression of an endogenoustissue morphogen, particularly OP-1 and other members of the largergenus of true tissue morphogens. The compounds thus identified haveutility both in vitro and in vivo. Useful compounds contemplated includeat least those that are capable of stimulating transcription and/ortranslation of the OP-1 gene, as well as compounds capable of inhibitingtranscription and/or translation of the OP-1 gene, via OP-1 non-codingDNA sequences resembling consensus sequences for Pax homeobox genes, inparticular, the Pax 6 or Pax 2 genes.

These and other objects and features of the invention will be apparentfrom the description, drawings and claims which follow.

SUMMARY OF THE INVENTION

The invention features compositions and methods for screening candidatecompounds for their ability to modulate the effective local or systemiclevels of endogenous morphogen, particularly OP-1, in an organism. Inone aspect, the method is practiced by: (I) incubating one or morecandidate compounds with cells transfected with a DNA sequence encodingat least a portion of a morphogen non-coding DNA sequence that isresponsive to a Pax homeobox gene and which is competent to act on andaffect expression of a reporter gene with which it is operativelyassociated; (2) measuring the level of reporter gene expression in thetransfected cell, and (3) comparing the level of reporter gene expressedin the presence of the candidate compound with the level of reportergene expressed in the absence of the candidate compound. The level of anexpressed reporter gene product in a given cell culture, or a change inthat level resulting from exposure to one or more compound(s) indicatesthat the compound can also modulate the level of the morphogen normallyassociated with the non-coding sequence. Specifically, an increase inthe level of reporter gene expression is indicative of a candidatecompound's ability also to increase morphogen expression in vivo.Similarly, a decrease in the level of reporter gene expression isindicative of a candidate compound's ability also to decrease orotherwise interfere with morphogen expression in vivo. The above methodis particularly useful for identifying compounds that are capable ofinfluencing Pax mediated OP-1 gene expression.

The methods of the invention can therefore be used to identify compoundsshowing promise as therapeutics for various in vivo and ex vivomammalian applications, as well as to identify compounds having numerousutilities. For example, compounds that modulate morphogen expression bystimulating Pax 2 or Pax 6 mediated transcription of a morphogen can beused in vivo to correct or alleviate a disease condition, to regeneratelost or damaged tissue, to induce cell proliferation anddifferentiation, and/or to maintain cell and tissue viability and/or adifferentiated phenotype in vivo or ex vivo. The compounds also can beused to maintain the viability of, and the differentiated phenotype of,cells in culture. The various in vivo, ex vivo, and in vitro utilitiesand applications of the morphogenic proteins described herein are welldocumented in the art. See, for example, US 92/01968 (WO 94/03200),filed Mar. 11, 1992; US 92/07358 (WO 93/04692), filed August 28; PCT US92/0743 (WO 93/05751), filed Aug. 28, 1992; US 93/07321 (WO 94/03200),filed Jul. 29, 1993; US 93/08808 (WO 94/06449), filed Sep. 16, 1993;US93/08885 (WO94/06420), filed Sep. 15, 1993, and U.S. Pat. No.5,266,683.

In another aspect, the invention further provides vectors and cellsuseful for morphogen, particularly OP-1, therapy. In one embodiment, theinvention features a vector having a reporter gene operativelyassociated with at least a portion of one or more OP-1 non-codingsequences responsive to Pax homeobox gene products. The OP-1 non-codingsequences comprise at least a first Pax responsive OP-1 modulatingelement which is responsive to a first Pax gene expression product. Inother embodiments, vectors further comprise a second non-coding sequencecomprising at least a second Pax-responsive OP-1 modulating elementwhich is responsive to a second Pax gene expression product; in suchembodiments, the first and second Pax gene expression product differ.OP-1 non-coding sequences which are Pax responsive OP-1 modulatingelements can be selected from nucleotides 1-3317 of SEQ ID No. 1. Alsoanticipated to be similarly useful are certain of the non-codingsequences of other species homologues of OP-1 and proteins closelyrelated to OP-1. For example, other non-coding DNA that is responsive toPax gene products or homologues thereof can be used to identifymodulators of specific morphogens, or other factors capable ofmodulating morphogen gene expression.

In another embodiment, the vector can include a non-coding OP-1-specificsequence selected from at least one of the following sequence segmentsof SEQ. ID No. 1 presented below, which defines approximately 3.3 Kb of5′ non-coding human genomic OP-1 sequence. Preferred vectors comprisesequence segments including nucleotides 1-3317, as well as shorterfragments of this region of DNA such as approximately nucleotides108-121, 139-154, 157-167, 365-378, 491-503, 598-613, 737-747, 891-903,994-1006, 1123-1140, 1144-1161, 1285-1297, 1750-1762, 2001-2023,2365-2378, 2931-2944 of SEQ. ID No. 1, including allelic, species andother sequence variants thereof. As base 2790 is the mRNA start site,other preferred sequences include approximately 2790-3317, representingtranscribed but not translated 5′ non-coding sequence and shorterfragments of this DNA region. Other preferred regions of the 5′non-coding region of SEQ. ID No. 1 include regions comprising a clusterof several Pax responsive elements, such as, for example, approximately1-2073, 1-1297, 1-2691, 1-378, 491-1006, 1750-2023, 1750-2378,1750-2691, 1750-2944. In certain embodiments, non-coding sequencescorrespond to part or all of SEQ. ID No. 2 and/or SEQ. ID No. 3,including allelic, species and other sequence variants thereof. In yetother embodiments, vectors comprise non-coding sequences correspondingto at least one, preferably between one and twelve and/or four or morefirst and second Pax-responsive OP-1 modulating elements, respectively.First Pax-responsive sites correspond approximately to bases 108-121,139-154, 157-167, 365-378, 497-511, 598-613, 1123-1140, 1144-1161,1285-1297, 1750-1762, 2001-2023, 2365-2378 and 2931-2944 of SEQ. IDNo. 1. Second Pax-responsive sites correspond approximately to bases491-503, 737-747, 891-903, and 994-1006 of SEQ. ID No. 1.

In another aspect, the invention provides a cell comprising a reportergene whose regulation is mediated by one or more of the Pax-responsiveOP-1 non-coding sequences defined above. In one embodiment, the cell istransfected with a reporter gene in operative association with at leastone Pax responsive site. In another embodiment, the present inventionprovides a cell comprising a transfected vector encoding a reporter geneoperatively associated with at least two DNA sequences, the firstcomprising at least part of a sequence selected from SEQ. ID No. 2 whilethe second comprises at least part of a sequence selected from SEQ. IDNo. 3, including allelic, species and other sequence variants of theforegoing. In yet another embodiment, cells of the present invention areco-transfected with expression vectors encoding Pax gene expressionproducts such as, for example, Pax 2 and/or Pax 6.

In another aspect, the invention provides kits useful in the designand/or identification of OP-1 expression modulating compounds. As usedherein a “kit” comprises a cell comprising a reporter gene in operativeassociation with an OP-1 non-coding DNA sequence and the reagentsnecessary for detecting expression of the reporter gene. The portion ofOP-1 noncoding DNA chosen can be any of the various sequences which havebeen described herein above.

Following this disclosure, medium flux screen assays, and kitstherefore, for identifying modulators of morphogen expression, such asOP-1 expression, are available. These compounds can be naturallyoccurring molecules, or they can be designed and biosyntheticallycreated using a rational drug design and an establishedstructure/function analysis methodology. The compounds can be aminoacid-based or can be composed in part or wholly of non-proteinaceoussynthetic organic molecules.

The OP-1 expression modulating compounds thus identified can be producedin reasonable quantities, including commercially significant quantities,using standard recombinant expression or chemical synthesis technologywell known and characterized in the art and/or as described herein. Forexample, automated means for the chemical synthesis of nucleic and aminoacid sequences are commercially available. Alternatively, promisingcandidate compounds can be modified using standard biological orchemical methodologies to, for example, enhance the binding affinity ofthe compound for a DNA element and the preferred candidate compoundderivative then can be produced in quantity.

Once a candidate compound has been identified and produced, it can befurther tested for its effect on OP-1 expression. For example, acompound which upregulates (increases) the production of OP-1 (e.g., anOP-1 agonist) in a kidney cell line is a candidate for systemicadministration. The candidate compound can be assayed in an animal modelto determine the candidate molecule's efficacy in vivo. For example, theability of a compound to upregulate levels of circulating OP-1 in vivocan be used to correct bone metabolism diseases such as osteoporosis(See, for example, PCT/US92/07932, above). Conversely, compounds whichdown regulate (decrease) the production of OP-1 (e.g., OP-1 antagonists)are also contemplated to be useful. Useful in vivo animal models forsystemic administration are disclosed in the art and below.

As is well known in the art, OP-1 is differentially expressed indifferent cell types. Accordingly, it is further anticipated that acandidate compound will have utility as an inducer of OP-1 expression inone cell type but not in another. Thus, the invention furthercontemplates testing a candidate compound for its utility in modulatingexpression of OP-1 in a tissue specific manner in vivo.

Thus, in view of this disclosure, one of ordinary skill in recombinantDNA and tissue culture techniques can design and construct appropriateDNA vectors and transfect cells with appropriate DNA sequences for usein the method according to the invention to assay for compounds whichmodulate the expression of OP-1. These identified compounds can be usedto modulate OP-1 production and its effective concentrations in both invivo and in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagrammatical illustration of the 5′ non-coding region ofthe OP-1 gene in operative association with a luciferase reporter gene.Certain preferred Pax consensus sites are depicted along the non-codingregion.

FIG. 1B is a graphical representation of the induction of luciferaseactivity in G401 human kidney cells transfected with OP-1 promoterdeletion constructs of (1A) in the presence or absence ofco-transfection with Pax 2 and/or Pax 6 expression constructs.

FIG. 2 (SEQ ID NOS: 2-3) shows the relative levels of luciferaseexpression from constructs containing 5′ non-coding regions of the OP-1gene in operative association with a luciferase reporter gene.

FIG. 3 shows the levels of luciferase expression in cells containingpAS3.3 and pAS3.3d.

DETAILED DESCRIPTION

As will be more fully described below, we have discovered specificregions in the OP-1 gene sequence useful in identifying moleculescapable of modulating OP-1 expression in vivo. We discovered DNAsequences responsible for the regulation of OP-1 gene expression bycloning and characterizing various truncated sequences isolated from the5′ non-coding sequences of the human OP-1 gene. The presence ofsequences defining Pax 2 or Pax 6 consensus binding sequences as definedherein in the OP-1 non-coding region, together with the observedmodulation of OP-1 gene expression mediated by Pax 2 and Pax 6 geneproducts, implicate these sequences, or variants thereof, as havingutility in a method for the screening of compounds for their ability tomodulate OP-1 expression. Moreover, Pax responsive sequences located in5′ non-coding sequences of other morphogen genes provide a means foridentifying compounds that modulate expression of other such morphogens.

List of Useful Terms and Definitions

As used herein, “morphogen” means the class of proteins typified byhuman osteogenic protein 1 (hOP-1). hOP-1 and functionally equivalentmorphogens are, as defined herein, dimeric proteins that induce orreinduce mammalian cells, particularly uncommitted progenitor cells, toundergo a fully integrated developmental cascade of cellular andmolecular events that culminates in the formation of fullydifferentiated, functional tissue of a type appropriate to the contextor local biological environment in which morphogenesis is induced,including any vascularization, connective tissue formation, enervationand the like characteristic of tissue naturally-occurring in such acontext. For example, if cells are stimulated by OP-1 in the context of,for example, bone, liver, nerve, tooth dentin, periodontal tissue,gastrointestinal tract lining tissue, the resulting cascade ofmorphogenesis culminates in the formation of new or regenerativedifferentiated tissue appropriate to that local environment.Morphogenesis therefore differs significantly from simple reparativehealing processes in which scar tissue (e.g., fibrous connective tissue)is formed and fills a lesion or other defect in differentiated,functional tissue.

Morphogens generally induce all of the following biological functions ina morphogenically permissive environment: stimulating proliferation ofprogenitor cells; stimulating the differentiation of progenitor cells;stimulating the proliferation of differentiated cells; and supportingthe growth and maintenance of differentiated cells. The term “progenitorcells” includes uncommitted cells, preferably of mammalian origin, thatare competent to differentiate into one or more specific types ofdifferentiated cells, depending on their genomic repertoire and thetissue specificity of the permissive environment in which morphogenesisis induced. Preferably, morphogenesis culminates in the formation ofdifferentiated tissue having structural and functional properties of atissue that occurs naturally in the body of a mammal. Morphogens furthercan delay or mitigate the onset of senescence- or quiescence-associatedLoss of phenotype and/or tissue function. Morphogens still further canstimulate phenotypic expression of differentiated cells, includingexpression of metabolic and/or functional, e.g., secretory, propertiesthereof. In addition, morphogens can induce redifferentiation oftransformed cells under appropriate environmental conditions. As notedabove, morphogens that induce proliferation and differentiation at leastof mammalian bone progenitor cells, and/or support the formation,growth, maintenance and functional properties of mammalian endochondralbone tissue, are representative.

A morphogen as isolated from natural sources in mature, biologicallyactive form is a glycosylated dimer typically having an apparentmolecular weight of about 30-36 kDa as determined by SDS-PAGE. Whenreduced, the 30 kDa protein gives rise to two glycosylated peptidesubunits having apparent molecular weights of about 16 kDa and 18 kDa.The reduced polypeptides themselves have no detectable morphogenicactivity. Glycosylation, however, is not required for biologicalactivity. The unglycosylated protein has an apparent molecular weight ofabout 27 kDa. When reduced, the 27 kDa protein gives rise to twounglycosylated polypeptides having molecular weights of about 14 kDa to16 kDa. The polypeptides which together form the biologically activedimer comprise at least six, preferably at least seven, positionallyconserved cysteine residues as set forth in U.S. Ser. No. 08/396,930 nowabandoned, the teachings of which have been incorporated herein byreference. As described above, particularly preferred sequences includethose comprising the C-terminal 96 or 102 amino acid sequences of DPP(from Drosophila), Vg1 (from Xenopus), Vgr-1 (from mouse), the OP-1 andOP2 proteins, proteins (see U.S. Pat. No. 5,011,691 and Oppermann etal., as well as the proteins referred to as BMP2, BMP3, BMP4 (seeWO88/00205, U.S. Pat. No. 5,013,649 and WO91/18098), BMP5 and BMP6 (seeWO90/11366, PCT/US90/01630), BMP8 and BMP9.

As stated above, the representative morphogen, for purposes of thepresent invention, comprises an OP-1 or an OP-1-related polypeptide.Sequences of useful OP-1 polypeptides are recited in U.S. Pat. Nos.5,011,691; 5,018,753 and 5,266,683; in Ozkaynak et al. (1990) EMBO J.2:2085-2093; and Sampath et al. (1993) Proc. Natl. Acad Sci. USA90:6004-6008. Additional useful sequences occur in the C-terminaldomains of DPP (from Drosophila), Vg1 (from Xenopus), 60A (fromDrosophila, see Wharton et al. (1991), Proc. Natl. Acad. Sci. USA88:9214-9218), Vgr-1 (from mouse), the OP-1 and OP2 proteins, (see U.S.Pat. No. 5,011,691 by Oppermann et al.), as well as the proteinsreferred to as BMP2, BMP3, BMP4 (see WO88/00205, U.S. Pat. No. 5,013,649and WO91/18098), BMP5 and BMP6 (see WO90/11366, PCT/US90/01630) and BMP8and 9. Each of the foregoing polypeptides, when oxidized and dimerized,is useful as a morphogen herein. Further, this family of morphogenicproteins includes longer forms of a given protein, as well asphylogenetic, e.g., species and allelic variants and biosyntheticmutants thereof, including addition and deletion mutants and variants,such as those which may alter the conserved C-terminal cysteineskeleton, provided that the alteration still allows the protein to forma dimeric species having a conformation capable of inducingmorphogenesis, e.g., endochondral bone formation when implanted in amammal in conjunction with a matrix permissive of bone morphogenesis. Inaddition, morphogens as defined herein can include forms having varyingglycosylation patterns and varying N-termini, can be naturally occurringor biosynthetically derived, and can be produced by expression ofrecombinant DNA in prokaryotic or eukaryotic host cells according toestablished techniques. The proteins are active either as homodimers orheterodimers.

As used herein, the terms “morphogen”, “bone morphogen”, “bonemorphogenic protein”, “BMP”, “osteogenic protein” and “osteogenicfactor” embrace the class of proteins a typified by human osteogenicprotein 1 (hOP-1). It will be appreciated by the artisan of ordinaryskill in the art, however, that OP-1 merely is representative of theTGF-β subclass of true tissue morphogens competent to act as osteogenicproteins, and is not intended to limit the description. Morphogenicprotein is generally understood to mean a protein which can induce thefull cascade of morphogenic events culminating in at least endochondralbone formation. Other known, and useful proteins include, OP2, OP3,BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11,BMP-12, BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8,GDF-9, GDF-10, GDF-11, GDF-12, Vg1, Vgr, 60A, DPP, NODAL, UNIVIN, SCREW,ADMP, NEURAL and morphogenically active amino acid variants thereof. Asdefined herein, morphogenic proteins include biologically active speciesvariants of any of these proteins, including conservative amino acidsequence variants, proteins encoded by degenerate nucleotide sequencevariants, and morphogenically active proteins sharing the conservedseven cysteine skeleton as defined herein and encoded by a DNA sequencecompetent to hybridize to a DNA sequence encoding an osteogenic proteindisclosed herein, including, without limitation, OP-1, BMP-5, BMP-6,BMP-2, BMP-4 or GDF-5, GDF-6 or GDF-7. Morphogenic proteins includethose sharing the conserved seven cysteine domain and sharing at least70% amino acid sequence homology (similarity) within the C-terminalactive domain, as defined herein. That is, particularly preferredmorphogenic proteins are those comprising an amino acid sequence havingat least 70% homology with the C-terminal 102-106 amino acids, definingthe conserved seven cysteine domain, of human OP-1 and related proteins.Certain preferred embodiments of the instant invention relate to themorphogenic protein, OP-1. “Amino acid sequence homology” is understoodherein to mean amino acid sequence similarity. Homologous sequencesshare identical or similar amino acid residues, where similar residuesare conservative substitutions for, or allowed point mutations of,corresponding amino acid residues in an aligned reference sequence.Thus, a candidate polypeptide sequence that shares 70% amino acidhomology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence.Examples of conservative variations include the substitution of onehydrophobic residue, such as isoleucine, valine, leucine or methionine,for another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic acid for asparticacid, or glutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid, provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide. Conservative substitutions typically include thesubstitution of one amino acid for another with similar characteristics,e.g., substitutions within the following groups: valine, glycine;glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamicacid; asparagine, glutamine; serine, threonine; lysine, arginine;phenylalanine, and tyrosine.

Naturally occurring proteins identified and/or appreciated herein to bemorphogenic proteins form a distinct subgroup within the looseevolutionary grouping of sequence-related proteins known as the TGF-βsuperfamily or supergene family. The naturally occurring morphogensshare substantial amino acid sequence homology in their C-terminalregions (domains). Morphogenic proteins comprise a pair of polypeptideswith amino acid sequences each comprising a sequence that shares adefined relationship with an amino acid sequence of a referencemorphogen. Herein, preferred morphogenic polypeptides share a definedrelationship with a sequence present in active human OP-1. However, anyone or more of the naturally occurring or biosynthetic sequencesdisclosed herein similarly could be used as a reference sequence.Preferred osteogenic polypeptides share a defined relationship with atleast the C-terminal six cysteine domain of human OP-1. Preferably,morphogenic polypeptides share a defined relationship with at least theC-terminal seven cysteine domain of human OP-1. That is, preferredpolypeptides in a dimeric protein with morphogenic activity eachcomprise a sequence that corresponds to a reference sequence or isfunctionally equivalent thereto.

Functionally equivalent sequences include functionally equivalentarrangements of cysteine residues disposed within the referencesequence, including amino acid insertions or deletions which alter thelinear arrangement of these cysteines, but do not materially impairtheir relationship in the folded structure of the dimeric morphogenprotein, including their ability to form such intra- or inter-chaindisulfide bonds as may be necessary for morphogenic activity.Functionally equivalent sequences further include those wherein one ormore amino acid residues differs from the corresponding residue of areference sequence, e.g., the C-terminal seven cysteine domain (alsoreferred to herein as the conserved seven cysteine skeleton) of humanOP-1, provided that this difference does not destroy morphogenicactivity. Accordingly, conservative substitutions of corresponding aminoacids in the reference sequence are preferred. Amino acid residues thatare conservative substitutions for corresponding residues in a referencesequence are those that are physically or functionally similar to thecorresponding reference residues, e.g., that have similar size, shape,electric charge, chemical properties including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al. (1978), 5 Atlas of ProteinSequence and Structure, Suppl. 3, ch. 22 (p. 354-352), Natl. Biomed.Res. Found., Washington, D.C. 20007, the teachings of which areincorporated by reference herein.

Publications disclosing these sequences, as well as their chemical andphysical properties, include: OP-1 and OP-2: U.S. Pat. No. 5,011,691,U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBO J. 9: 2085-2093;OP-3: WO94110203 PCT US93/10520); BMP2, BMP3, BMP4: WO88/00265, Wozneyet al. (1988) Science 242: 1528-1534); BMP5 and BMP6: Celeste et al(1991) PNAS 87: 9843-9847; Vgr-1: Lyons et al. (1989) PNAS 86:4554-4558; DPP: Padgett et al. (1987) Nature 325: 81-84; Vg-1: Weeks(1987) Cell 51: 861-867; BMP-9: WO95/33830(PCT/US95/07084); BMP10:WO94/26893 (PCT/US94/05290); BMP-11: WO94/26892 (PCT/US94/05288); BMP12:WO95/16035 (PCT/US94/14030); BMP-13: WO95/16035 (PCT/US94/14030); GDF-1:WO92/00382 (PCT/US91/04096) and Lee et al. (1991) PNAS 88: 4250-4254;GDF-8: WO94/21681 (PCT/US94/03019); GDF-9: WO94/15966 (PCT/US94/00685);GDF-10: WO95/10539 (PCT/US94/11440); GDF-11: WO96/01845(PCT/US95/08543); BMP-15: WO96/36710 (PCT/US96/06540); MP121: WO96/01316(PCT/EP95/02552); GDF-5 (CDMP-1, MP52): WO94/15949 (PCT/US94/00657) andWO96/14335 (PCT/US94/12814) and WO93/16099 (PCT/EP93/00350); GDF(CDMP-2, BMP13): WO95/01801 (PCT/US94/07762) and WO96/14335 andWO95/10635 (PCT/US94/14030); GDF-7 (CDMP-3. BMP12): WO95/10802(PCT/US94/07799) and WO95/10635 (PCT/US94/14030). In another embodiment,useful proteins include biologically active biosynthetic constructs,including novel biosynthetic morphogenic proteins and chimeric proteinsdesigned using sequences from two or more known morphogens. See also thebiosynthetic constructs disclosed in U.S. Pat. No. 5,011,691, thedisclosure of which is incorporated herein by reference (e.g., COP-1,COP-3, COP-4, COP-5, COP-7, and COP-16).

As earlier stated, morphogenic proteins contemplated herein includethose in which the amino acid sequences comprise a sequence sharing atleast 70% amino acid sequence homology or “similarity”, and preferably80% homology or similarity, with a reference morphogenic proteinselected from the foregoing naturally occurring proteins. Preferably,the reference protein is human OP-1, and the reference sequence thereofis the C-terminal seven cysteine domain present in osteogenically activeforms of human OP-1. A polypeptide suspected of being functionallyequivalent to a reference morphogen polypeptide is aligned therewithusing the method of Needleman, et al. (1970) J. Mol. Biol. 48:443-453,implemented conveniently by computer programs such as the Align program(DNAstar, Inc.). As noted above, internal gaps and amino acid insertionsin the candidate sequence are ignored for purposes of calculating thedefined relationship, conventionally expressed as a level of amino acidsequence homology or identity, between the candidate and referencesequences. “Amino acid sequence homology” is understood herein toinclude both amino acid sequence identity and similarity. Homologoussequences share identical and/or similar amino acid residues, wheresimilar residues are conservative substitutions for, or “allowed pointmutations” of, corresponding amino acid residues in an aligned referencesequence. Thus, a candidate polypeptide sequence that shares 70% aminoacid homology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence. Ina currently preferred embodiment, the reference sequence is OP-1.Morphogenic proteins useful herein accordingly include allelic,phylogenetic counterpart and other variants of the preferred referencesequence, whether naturally-occurring or biosynthetically produced(e.g., including “muteins” or “mutant proteins”), as well as novelmembers of the general morphogenic family of proteins, including thoseset forth and identified above. Certain particularly preferredmorphogenic polypeptides share at least 60% amino acid identity with thepreferred reference sequence of human OP-1, still more preferably atleast 65% amino acid identity therewith.

As noted above, certain currently preferred morphogenic polypeptidesequences have greater than 60% identity, preferably greater than 65%identity, with the amino acid sequence defining the preferred referencesequence of hOP-1. These particularly preferred sequences includeallelic and phylogenetic counterpart variants of the OP-1 and OP-2proteins, including the Drosophila 60A protein. Accordingly, preferredmorphogenic proteins include active proteins comprising pairs ofpolypeptide chains within the generic amino acid sequence hereinreferred to as “OPX” (see below and SEQ ID NO: 4), which defines theseven cysteine skeleton. As described therein, each Xaa at a givenposition independently is selected from the residues occurring at thecorresponding position in the C-terminal sequence of mouse or human OP-1or OP-2.

Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp Xaa Asp Trp1               5                   10                  15 Xaa Ile AlaPro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro    20                  25                  30                  35 LeuXaa Ser Xaa Met Asn Ala Thr Asn His Ala Ile Xaa Gln Xaa Leu Val His Xaa        40                  45                  50                  55Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala            60                  65                  70 Xaa Ser Val LeuTyr Xaa Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys Xaa Arg75                  80                  85                  90 Asn MetVal Val Xaa Ala Cys Gly Cys His         95                  100

wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg); Xaa atres: 11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res. 19=(Ileor Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala or Ser); Xaaat res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa at res.41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Ser orThr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaa atres. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Glnor Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaaat res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr orHis); and Xaa at res. 97=(Arg or Lys).

In yet other preferred embodiments, morphogenic protein as contemplatedherein can be further defined by a generic amino acid sequence. Forexample, SEQ. ID No. 5 and No. 6 disclosed herein represent compositeamino acid sequences of the following proteins: human OP-1, human OP-2,human OP-3, human BMP-2, human BMP-3, human BMP-4, human BMP-5, humanBMP-6, human BMP-8, human BMP-9, human BMP10, human BMP-11, Drosophila60A. Xenopus Vg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), humanCDMP-2 (mouse GDF-6, human BMP-13), human CDMP-3 (mouse GDF-7, humanBMP-12), mouse GDF-3, human GDF-1, mouse GDF-1, chicken DORSALIN, dpp,Drosophila SCREW, mouse NODAL, mouse GDF-8, human GDF-8, mouse GDF-9,mouse GDF-10, human GDF-11, mouse GDF-11, human BMP-15, and rat BMP3b.SEQ. ID NO: 5 accommodates the C-terminal six cysteine skeleton and,SEQ. ID NO: 6 accommodates the seven cysteine skeleton.

    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa SEQ.ID NO: 5       1               5                  10                  15Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Xaa            20                  25                  30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa    35                  40                  45                  50 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa                55              60                      65 Xaa Xaa XaaXaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa        70                  75                  80 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Cys Xaa Cys Xaa 85                  90                  95

wherein each Xaa is independently selected from a group of one or morespecified amino acids defined as follows: “Res.” means “residue” and Xaaat res. 1=(Phe, Leu or Glu); Xaa at res. 2=(Tyr, Phe, His, Arg, Thr,Lys, Gln, Val or Glu); Xaa at res. 3 (Val, Ile, Leu or Asp); Xaa at res.4=(Ser, Asp, Glu, Asn or Phe); Xaa at res. 5=(Phe or Glu); Xaa at res.6=(Arg, Gln, Lys, Ser, Glu, Ala or Asn); Xaa at res. 7=(Asp, Glu, Leu,Ala or Gln); Xaa at res. 8=(Leu, Val, Met, Ile or Phe); Xaa at res.9=(Gly, His or Lys); Xaa at res. 10=(Trp or Met); Xaa at res. 11=(Gln,Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser,Lys, Arg, Glu or His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ileor Val); Xaa at res. 15=(Ile or Val); Xaa at res. 16=(Ala, Ser, Tyr orTrp); Xaa at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, His or Lys);Xaa at res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gln, His, Glu, Asp,Leu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at res. 23=(Tyr,Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu, Phe or Arg); Xaaat res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln or Gly); Xaa atres. 28=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gln); Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Phe, Gln or Leu); Xaa at res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res. 32=(Pro, Ser, Ala orVal); Xaa at res. 33=(Leu, Met, Glu, Phe or Val); Xaa at res. 34=(Asn,Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu,Asp, Thr, Leu, Lys, Gln or His); Xaa at res. 36=(Tyr, His, Cys, Ile,Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe,Val, Gly or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly,Met, Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa atres. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys, Val,Thr or Gln); Xaa at res. 42=(His, Tyr or Lys); Xaa at res. 43=(Ala, Thr,Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaaat res. 45=(Val, Leu, Met, Ile or His); Xaa at res. 46=(Gln, Arg orThr); Xaa at res. 47=(Thr, Ser, Ala, Asn or His); Xaa at res. 48=Cu, Asnor Ile); Xaa at res. 49=(Val, Met, Leu, Pro or Ile); Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gln); Xaa at res. 51=(Phe, Leu, Ser, Asn,Met, Ala, Arg, Glu, Gly or Gln); Xaa at res. 52=(Ile, Met, Leu, Val,Lys, Gln, Ala or Tyr); Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala,Gln, Gly, Leu or Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaaat res. 55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gln, Pro or His);Xaa at res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaaat res. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res. 58=(Pro, Gly,Ser, Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg orGly); Xaa at res. 60=(Pro, Ala, Val, Thr or Ser); Xaa at res. 61=(Cys,Val or Ser); Xaa at res. 63=(Ala, Val or Thr); Xaa at res. 65=(Thr, Ala,Glu, Val, Gly, Asp or Tyr); Xaa at res. 66=(Gln, Lys, Glu, Arg or Val);Xaa at res. 67=(Leu, Met, Thr or Tyr); Xaa at res. 68=(Asn, Ser, Gly,Thr, Asp, Glu, Lys or Val); Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaaat res. 70=(Ile, Thr, Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr,Asn or Gly); Xaa at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr,Phe, Arg, Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile,Lys, Gln or Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res.77=(Asp, Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res. 78=(Ser,Asn, Asp, Tyr, Ala, Gly, Gln, Met, Glu, Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at res. 80=(Asn,Lys, Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at res. 81=(Val, Ile, Thror Ala); Xaa at res. 82=(Ile, Asn, Val, Leu, Tyr, Asp or Ala); Xaa atres. 83=(Leu, Tyr, Lys or Ile); Xaa at res. 84=(Lys, Arg, Asn, Tyr, Phe,Thr, Glu or Gly); Xaa at res. 85=(Lys, Arg, His, Gln, Asn, Glu or Val);Xaa at res. 86=(Tyr, His, Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln,Pro or Lys); Xaa at res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa atres. 89=(Met or Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys);Xaa at res 91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gln, Asp, Glu,Val, Ala, Ser or Thr); Xaa at res. 93 (Ala, Ser, Glu, Gly, Arg or Thr);Xaa at res. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu orSer). Further, after res. 53 in rBMP3b and mGDF-10 there is an Ile;after res. 54 in GDF-1 there is a T; after res. 54 in BMP3 there is a V;after res. 78 in BMP-8 and Dorsalin there is a G; after res. 37 inhGDF-1 there is Pro, Gly, Gly, Pro.

SEQ ID NO: 6 includes all of SEQ ID NO: 5 and in addition includes thefollowing sequence (SEQ ID NO: 7) at its N-terminus:

Cys Xaa Xaa Xaa Xaa SEQ ID NO: 7   1               5

Accordingly, beginning with residue 6, each “Xaa” in SEQ. ID No. 6 is aspecified amino acid defined as for SEQ. ID No. 5, with the distinctionthat each residue number described for SEQ. ID No. 5 is shifted by fivein SEQ. ID No. 6. Thus, “Xaa at res. 1=(Tyr, Phe, His, Arg, Thr, Lys,Gln, Val or Glu)” in SEQ. ID No. 5 refers to Xaa at res. 6 in SEQ. IDNo. 6. In SEQ. ID No. 6, Xaa at res. 2=(Lys, Arg, Gln, Ser, His, Glu,Ala, or Cys); Xaa at res. 3=(Lys, Arg, Met, Lys, Thr, Leu, Tyr, or Ala);Xaa at res. 4=(His, Gln, Arg, Lys, Thr, Leu, Val, Pro, or Tyr); and Xaaat res. 5=(Gln, Thr, His, Arg, Pro, Ser, Ala, Gln, Asn, Tyr, Lys, Asp,or Leu).

Still further, morphogenically active proteins have polypeptide chainswith amino acid sequences comprising a sequence encoded by a nucleicacid that hybridizes, under low, medium or high stringency hybridizationconditions, to DNA or RNA encoding reference morphogen sequences, e.g.,C-terminal sequences defining the conserved seven cysteine domains ofOP-1, OP-2, BMP2, BMP4, BMP5, BMP6, 60A, GDF3, GDF6, GDF7 and the like.As used herein, high stringency hybridization conditions are defined ashybridization according to known techniques in 40% formamide, 5×SSPE, 5×Denhardt's Solution, and 0.1% SDS at 37° C. overnight, and washing in0.1×SSPE, 0.1% SDS at 50° C. Standard stringency conditions are wellcharacterized in commercially available, standard molecular cloningtexts. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed.,ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor LaboratoryPress: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984): Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); and B. Perbal, APractical Guide To Molecular Cloning (1984).

As used herein, “gene expression” is understood to refer to theproduction of a gene product encoded by a DNA sequence of interest,including the transcription of the DNA sequence and/or translation ofthe mRNA transcript. It is understood that gene expression in thecontext of the present invention is not necessarily a consequence ofdirect interaction of specific proteins or factors with specific DNAsequences of the present invention, but that the reporter gene productis produced as a consequence of an inductive effect that is mediated orinfluenced by the presence and/or expression of Pax genes in the cellsof the invention.

As used herein, “operative association” relates to a fusion of thedescribed non-coding DNA sequences with a reporter gene in such areading frame as to be co-transcribed, or at such a relative positioningas to be competent to modulate expression of the reporter gene.

As used herein, “vector” is understood to mean any nucleic acidcomprising a nucleotide sequence of interest and competent to beincorporated into a host cell and capable of functional transcription.Such vectors include linear or circular nucleic acids, plasmids,phagemids, cosmids, YACs (yeast artificial chromosomes) and the like.Vector as used herein can be used to construct a test cell, or constructDNAs, cells and/or viruses suitable for gene therapy applications.

As used herein, “non-coding sequence” or “non-coding DNA” includes DNAsequences that are not transcribed into RNA sequence, and/or RNAsequences that are not translated into protein. This category of“non-coding sequence” has been defined for ease of reference in theapplication, and includes sequences occurring 5′ to the ATG site of theOP-1 gene at nucleotide 3318 of SEQ. ID No. 1. Further, as used herein“5′ non-coding sequence” or “5′ non-coding DNA” define a gene locusupstream of or 5′ to the translation start site of a reporter gene. Incontrast, coding sequence means a translated and/or transcribed DNAsequence encoding a reporter gene or morphogenic protein as definedherein.

As used herein, an “OP-1-specific” or “morphogen-specific” non-codingsequence is understood to define a non-coding sequence that liescontiguous to an OP-1-specific or morphogen specific coding sequence ata gene locus under naturally-occurring conditions.

“Responsive,” as defined herein, is not limited to direct interactionbetween elements of the invention (e.g. Pax responsive OP-1 modulatingelement and Pax gene products) but includes indirect effects on genetranscription or translation, for example, that are mediated orinfluenced by the Pax gene products.

As used herein, a “Pax responsive OP-1 modulating element” is a DNAelement that is responsive to the presence of a Pax gene and/orexpression of a Pax gene product. That is, expression of a geneoperatively associated therewith is modulated when present in a celltogether with DNA encoding a Pax gene. For example, when a reporter geneand Pax gene are co-transfected into a host cell, reporter geneexpression is modulated by the presence and/or expression of anendogenous Pax gene. As discussed herein, it is contemplated that a Paxresponsive modulating element(s) can be located in the 5′ non-codingsequences of any morphogen as defined herein. Thus, for purposes of thepresent invention, a Pax responsive morphogen modulating elementcomprises at least all of the generic features (structural, functional,chemical) associated with a Pax responsive OP-1 modulating element, andfurther includes allelic, species and mutant variants thereof. A “Paxgene product” is a nucleic acid or protein product encoded by a genewhich is a member of the Pax family of genes.

“Pax genes” encode a family of developmentally regulated transcriptionfactors that have been implicated in a number of human and murinecongenital disorders, as well as in tumorigenesis. These genes, alsocharacterized as “paired box genes,” are defined by the presence of anevolutionarily conserved DNA binding domain, termed the “paired domain”.As demonstrated below in Example 2, Pax genes and/or their expressionand/or transcription products are morphogen modulators as that group ofcompounds is defined herein.

Paired box genes were first identified in Drosophila as a family ofrelated genes encoding a 128-amino acid DNA binding domain, and areexpressed in temporally and spatially restricted patterns duringdevelopment. The phenotypes associated with Pax gene mutationsdemonstrate that these gene products are critical during organogenesis.Missense mutation within the paired domain of Pax genes have beenassociated with congenital disorders in both mouse and man. Also, Paxgenes have been demonstrated to have oncogenic potential, and, forexample, a translocation involving the paired box portion of Pax 3 hasbeen associated with a human tumor.

The consensus binding sequences for Pax 6 and Pax 2 paired domains arevery similar. The Pax 6 consensus spans 20 base pairs and shares acentral 10-base pair region of homology with the Pax 2 consensusconsisting of TCACGC-TGA (SEQ ID NO: 15), where the dash indicates anucleotide difference between the two sequences in this central basepair region (Epstein et al. (1994) J. Biol. Chem. 269:8355-8361, thedisclosure of which is incorporated by reference herein).

The respective purified paired domain proteins exhibit high affinity fortheir respective consensus sequences. The Pax-6 paired domain has beenshown to bind to this consensus sequence as a monomer. The paireddomain, when binding to such a DNA sequence, adopts an α-helicalconformation and contacts residues within a large span of the DNAmolecule.

As used herein, a “Pax 2 consensus binding sequence” or “Pax 2 consensusbinding element” is a nucleotide sequence which has been shown to bebound by the DNA binding protein Pax 2. The consensus sequence of thePax 2 binding site has been determined by homology to be: G T C A C/T GC G/A T G A, as depicted in SEQ. ID No. 3. (Epstein et al., (1994) J.Biol. Chem. 269:8355-8361). A “Pax 6 consensus binding sequence” or “Pax6 consensus binding element” is a nucleotide sequence which has beenshown to be bound by the DNA binding protein Pax 6. The consensussequence of the Pax 6 binding site has been determined by homology to beT T C A C G C A/T T G/C A N T G/T A/C N T/C, as depicted in SEQ. ID No.2 where N=A, G, C or T. (Epstein et al. (1994) J. Biol. Chem.269:8355-8361.) Another suitable consensus sequence for purposes of thepresent invention is A--TTCACGCATGA-T (SEQ ID NO: 8) or TTCACGCATGA (SEQID NO: 9), wherein the dashes indicate that any nucleotide can reside atthat locus.

“Pax 2 or Pax 6 mediated transcription or expression” meanstranscription or expression of a reporter gene operatively associatedwith a Pax responsive modulating element, or functional equivalentthereof, as described elsewhere herein. Such transcription or expressionoccurs in the presence of Pax genes and/or Pax gene expression products,but their presence is not required. “Pax 2 or Pax 6 mediatedtranscription or expression” can involve, for example, the directphysical interaction of Pax 2 or Pax 6 gene products with the Pax 2 orPax 6 consensus binding sequences, as defined herein above.Alternatively, “Pax mediated transcription or expression” can bemodulated by compounds that can mimic the transcription or expressionevents otherwise associated with Pax family proteins. Such compounds arecontemplated herein generally as morphogen modulators, and morespecifically as Pax analogs.

As discussed elsewhere herein, “allelic, species and other sequencevariants thereof” includes point mutations, insertions and deletionssuch as those that are naturally occurring or which can be geneticallyengineered into an OP-1 non-coding DNA sequence using routine methodsand which do not substantially affect the regulation of a reporter geneby the OP-1 non-coding sequence. For example, one of ordinary skill inthe art can use site directed mutagenesis to modify, as by deletion, forexample, one or more of the OP-1 non-coding sequences described hereinwithout substantially affecting the regulation of OP-1 or a reportergene by the modification. Such modifications are considered to be withinthe scope of the disclosure provided herein.

As discussed elsewhere herein, “analog” or “variant” is intended to meana DNA or a protein which mimics or performs similar functions relativeto a naturally-occurring or representative DNA or protein. For example,a DNA that is responsive to Pax 2 or Pax 6 in the same manner as thosedescribed herein, yet are structurally different therefrom, areconsidered DNA analogs or DNA variants within the scope of thisinvention. Similarly, any expression product which can modulate OP-1expression in the same manner as Pax 2 and/or Pax 6 as disclosed herein,are considered Pax 2 and/or Pax 2 analogs or variants within the scopeof this invention.

“Co-transfection” refers to the simultaneous or sequential transfectionof two or more vectors into a given cell.

Where a cell line is to be established, particularly where thetransfected DNA is to be incorporated into the cell's genome, lines thatcan be immortalized are especially desirable. As used herein,“immortalized” cell lines are viable for multiple passages withoutsignificant reduction in growth rate or protein production. It iscontemplated that the cells of the present invention have utility forcell mediated gene therapy, where cells producing high levels of areporter gene such as OP-1 are desired. It is further contemplated thatthe Pax consensus binding sequences or other useful sequences can bealtered or adapted for viral mediated gene therapy using routinemethods.

The Pax genes and/or gene products as used herein are capable ofstimulating the transcription of a morphogen gene and are herein alsoreferred to as “morphogen stimulators”, “morphogen modulating compounds”or “OP-1 modulating compounds.”

A “candidate compound” is any test substance that can be used to treatthe cells of the present invention in vitro, in vivo or ex vivo todetermine their usefulness as OP-1 modulating compounds. Accordingly,and in keeping with the earlier definition, a candidate compound whichcan modulate OP-1 expression in the manner of Pax 2 and/or Pax 6 asdisclosed herein, is also a morphogen stimulator as well as a Pax 2and/or Pax 6 analog. Conversely, a candidate compound which actscontrary to Pax 2 and/or Pax 6 as disclosed herein, is a morphogensuppressor or Pax 2/Pax 6 antagonist.

The OP-1 Upstream Region Contains a Silencer of Gene Expression

The presence of a sequence within the OP-1 upstream region that reducesOP-1 gene expression (a silencer fragment) was identified in expressionassays described below in Example 9. The approximately 0.1 kbAatII-PvuII fragment (approximately nucleotides 2606-2690 of SEQ IDNO: 1) reduces OP-1 expression by approximately three fold (betweenapproximately two fold and five fold in the experiments described inExample 9).

In a preferred embodiment of the invention, expression of OP-1 isincreased by removing the silencer fragment from the OP-1 upstreamregion. In one embodiment, the silencer fragment is removed from aplasmid or other recombinant construct containing the OP-1 gene. In analternative embodiment, a recombinant cell line is made wherein one orboth genomic copies of the OP-1 gene are modified to remove the silencerfragment, using methods known in the art.

In another embodiment of the invention, the silencer fragment is used tomodulate the expression of another (non-OP-1) gene. One or more copiesof the silencer fragment are inserted in the promoter region of the genein order to reduce expression. The silencer fragment is inserted intothe promoter region of a gene that is contained in a plasmid, arecombinant vector, or in the genome itself.

The silencer fragment contained within the AatII-PvuII fragment can bemore precisely identified by standard deletion analysis of the fragmentusing the expression assay described in Examples 2 and 9. Truncatedsilencer fragments, identified by a further deletion analysis, are alsouseful in the invention.

A naturally occurring sequence variant of the silencer fragment (or of atruncated silencer fragment), or a nucleic acid which hybridizes to thesilencer fragment (or to a truncated siencer fragment) under highstringency hybridization conditions, is also useful in the invention.

Exemplary Cells, Vectors, Reporter Genes and Assays for Use in ScreeningCompounds Which Modulate OP-1 Gene Expression

A. Useful Cells

Any eukaryotic cell, including an immortalized cell line suitable forlong term culturing conditions, is contemplated to be useful for themethods and cells of the invention. Useful cells should be easy totransfect, are capable of stably maintaining foreign DNA with anunrearranged sequence, and have the necessary cellular components forefficient transcription and translation of the protein, including anyelements required for post-translational modification and secretion, ifnecessary. Where the cell is to be transfected with a non-dominatingselection gene, the cell genotype preferably is deficient for theendogenous selection gene. Preferably, the cell line also has simplemedia composition requirements, and rapid generation times. Useful celllines are mammalian cell lines, including myeloma, HeLa, fibroblast,embryonic and various tissue cell lines, e.g., kidney, liver, lung andthe like. The cells may be derived from tissue or subcultured fromestablished cell lines. As used herein, “derived” means the cells arefrom the cultured tissue itself or are a cell line whose parent cellsare of the tissue itself. Cell lines particularly useful in practicingthe present invention are, for example, Y79 human retinoblastoma cells,G401 human kidney cells, ROS human osteoblastic cells, MCF-7 humanmammary cancer cells and LLCBK1 porcine proximal tubule cells. A largenumber of cell lines now are available through the American Type CultureCollection (Rockville, Md.) or through the European Collection of AnimalCell Cultures (Porton Down, Salisbury, SP4 0JG, U.K.)

Where the expression of a reporter gene that is controlled by non-codingsequences of the morphogen OP-1 is to be analyzed, particularly usefulcells and cell lines are envisioned to include eukaryotic, preferablymammalian cells of a tissue and cell type known to express OP-1 and/orclosely related proteins. See, for example, Ozkaynak, et al. (1991),Biochem. Biophys. Res. Commun. 179: 116-123 for a detailed descriptionof tissues known to express OP-1. Such cells, include, withoutlimitation, cells of uro-genital cell origin, including kidney, bladderand ovary cells, lung, liver, bone, nerve, mammary gland and cardiaccells, cells of gonadal origin, cells of gastrointestinal origin, glialcells and other cell lines known to express endogenous genes encodingmorphogenic proteins. Preferred cell lines are of epithelial origin.

Cell cultures of kidney, adrenals, urinary bladder, brain, or otherorgans, can be prepared as described widely in the literature. Forexample, kidneys can be explanted from neonatal or new born or young oradult rodents (mouse or rat) and used in organ culture as whole orsliced (1-4 mm) tissues. Primary tissue cultures and established celllines, also derived from kidney, adrenals, urinary, bladder, brain,mammary, or other tissues can be established in multiwell plates (6 wellor 24 well) according to conventional cell culture techniques, and arecultured in the absence or presence of serum for a period of time (1-7days). Cells can be cultured, for example, in DMEM containing 10% fetalcalf serum or in serum-deprived medium or in defined medium (e.g.,containing insulin, transferrin, glucose, albumin, or other growthfactors). Test compounds are added to the cultured cells and OP-1biosynthesis monitored and measured at various time points using themethods described previously herein. Suitable cell lines include celllines that have been shown to contain high levels of OP-1 mRNA,indicating that the OP-1 promoter is active in the cells. Cells andtheir culture fluids are assayed using techniques well known in the art,either for mRNA levels, using Northern blot analysis and OP-1 mRNAspecific probes, or for protein levels, using OP-1 specific antibodies.For example, OP-1 protein can be measured on a tissue section or celldirectly using standard immunofluorescence techniques or in culturefluids and body fluids using a sandwich immunoassay. Other methods fordetecting, measuring and purifying proteins are well know in the art.

Alternatively, in one aspect the present invention may be practiced inyeast cells such as S. cerevisiae to identify or further characterizeprotein-protein interactions of the identified intracellular OP-1modulating factors of the present invention. For example, the yeast twohybrid system, as described in Kalpana et al. (1993) 90 Proc. Natl.Acad. Sci. USA. (22): 10593-10597 and already well known to thoseskilled in the art, is useful for delineating domains or criticalresidues for an interaction between two proteins, for example, a proteinX and a protein Y. Briefly, hybrid genes are constructed for expressionin yeast comprising (a) a DNA binding domain fused to a protein X and(b) an activator domain fused to a protein Y. Transcriptional activationin these experiments is only restored when protein X interacts withprotein Y leading to close contact of both the binding domain and theactivation domain. Transcription activation is then monitored by areporter gene resident in the yeast strain.

B. Exemplary Vectors/Vector Construction Considerations

Useful vectors for use in the invention include, but are not limited toplasmids, cosmids, phagemids, yeast artificial chromosomes or otherlarge vectors. Vectors that can be maintained within the nucleus orintegrated into the genome by homologous recombination are also useful.

Selected portions of noncoding OP-1 sequence can be cloned into a usefulvector using standard molecular cloning techniques, as exemplifiedbelow. Restriction endonuclease sites will be utilized when possible,and can be engineered into the sequence when needed. Restrictionendonuclease sites can be engineered into the non-coding sequence usingthe common techniques such as site directed mutagenesis and PCR withprimers including the desired restriction endonuclease site.

Also envisioned is a nucleic acid construct comprising a small fragmentof 5′ non-coding OP-1 sequence in combination with additional conservedelements such as one or more Pax 6 binding sequences and/or Pax 2binding sequences in operative association with a reporter gene.

A range of useful 5′ non-coding fragments is provided herein, and aswill be apparent to those of ordinary skill in the art, smallerfragments of OP-1 sequence also are useful. Such smaller fragments canbe identified by deleting bases from one or both ends of the provided 5′noncoding fragments, using techniques that are well known in the art andtesting the truncated constructs for their ability to modulate reportergene expression. In this way, the shortest modulating sequences can beidentified.

C. Transfection Considerations

Any routine method for incorporating nucleic acids into cells ofinterest is contemplated in the method of the invention. For example,calcium phosphate (CaPO₄), followed by glycerol shock is a standardmeans used in the art for introducing vectors, particularly plasmid DNAinto mammalian cells. A representative method is disclosed in Cockett etal., (1990) Biotechnology 8:662-667, incorporated herein by reference.Other methods that may be used include electroporation, protoplastfusion (particularly useful in myeloma transfections), microinjections,lipofections and DEAE-dextran mediated uptake. Methods for theseprocedures are described in F. M. Ausubel, ed., Current Protocols inMolecular Biology, John Wiley & Sons, New York (1989). An importantaspect of the invention is the DNA sequences with which the cell istransfected, rather than the mechanical or chemical process by which theDNA incorporation is accomplished.

As will be appreciated by those having skill in the art, optimal DNAconcentrations per transfection and other standard conditions will varyaccording to the transfection protocol. For calcium phosphatetransfection, for example, preferably 5-10 μg plasmid DNA per plasmidtype is transfected. In addition, the DNA to be transfected preferablyis essentially free of contaminants that may interfere with DNAincorporation. A standard means used in the art for purifying DNA is byethidium bromide banding.

D. Exemplary Reporter Genes

Reporter genes are characterized as being easy to transfect into asuitable host cell, easy to detect using an established assay protocol,and genes whose expression can be tightly regulated. Other reportergenes contemplated to have utility include, without limitation, theluciferase gene, the Green Fluorescent Protein (GFP) gene, human growthhormone, GAL4 and β-galactosidase.

A well-recognized reporter system is the firefly luciferase reportersystem. See, for example Gould, S. J., and Subramani, S. (1988) Anal.Biochem., 7:404-408 for a description of the reporter gene and generalmethodology. The luciferase assay is fast and has increased sensitivity.Further, the half-life of luciferase protein is short and thereforeallows for accurate kinetic studies of luciferase production. The systemalso is particularly useful in bulk transfections or if the promoter ofinterest is weak. In this assay transfected cells are grown understandard conditions, and when cultured under assay conditions both ATPand the substrate luciferin is added to the cell lysate. The enzymeluciferase catalyzes a rapid, ATP dependent oxidation of the substratewhich then emits light. The total light output is measured using aluminometer according to manufacturer's instructions (e.g., Promega) andis proportional to the amount of luciferase present over a wide range ofenzyme concentrations. For example, a vector such as pGL2-LUC (Promega)is particularly useful.

A second well-known reporter system is based on immunologic detection ofhGH, it is quick and easy to use. (Selden, R., Burke-Howie, K. Rowe, M.E., Goodman, H. M., and Moore, D. D. (1986), Mol. Cell Biol.,6:3173-3179 incorporated herein by reference). hGH is assayed in themedia, rather than in cell extracts. This allows direct monitoring overby a single population of transfected cells over time.

Additional useful reporter genes are any well characterized genes theexpression of which is readily assayed, and examples of such reportergenes can be found in, for example, F. A. Ausubel et al., Eds., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, (1989). Aswill be appreciated by those having ordinary skill in the art, thelisted reporter genes are only a few of the possible reporter genes, andit is only for ease of description that all available reporter genes arenot listed. It will be apparent to those of ordinary skill in the artthat genes encoding other detectable proteins of interest, such as thegene encoding human OP-1 shown in SEQ. ID No. 1, or other morphogenwhose enhanced expression is desirable, is also within the scope of asuitable reporter gene.

As indicated above and as will be appreciated by those having ordinaryskill in the art, particular details of the conventional means fortransfection, expression, and assay of recombinant genes are welldocumented and are understood by those having ordinary skill in the art.The instant invention enables and discloses vectors, cells and a methodfor screening compounds to determine the capability of compounds tomodulate the expression of OP-1 via the non-coding sequences of the OP-1genomic DNA.

Further details on the various technical aspects of each of the stepsused in recombinant production of foreign genes in mammalian expressionsystems can be found in a number of texts and laboratory manuals in theart, such as, for example, F. M. Ausubel et al., Ed., Current Protocolsin Molecular Biology, John Wiley & Sons, New York, (1989).

In view of this disclosure and the examples provided below, a method foridentifying molecules which can affect OP-1 expression in a particularcell type in vivo now is provided.

EXAMPLE 1 Cloning of Human OP-1 Gene Non-Coding Sequences

Human OP-1 upstream non-coding sequence was obtained by screening thehuman genomic library, HL 1067J. (Clontech). The library was screened byan initial plating of 750,000 plaques (approximately 50,000plaques/plate). Hybridizations were done in 40% formamide, 5×SSPE,5×Denhardt's solution, and 0.1% SDS at 37° C. using a ³²P-labeled probemade from a human 0.47 kb EcoRI OP-1 cDNA fragment containing mainly 5′non-coding and exon 1 sequences. Nonspecific counts were removed in0.1×SSPE, 0.1% SDS by shaking at 50° C.

A 7 kb EcoRI fragment from the human genomic clone, lambda Ö3, wasisolated and sequenced and contained 5 kb of OP-1 upstream non-codingsequence.

All sequencing was done according to Sanger et al. (1977) Proc. Natl.Acad. Sci. 14:5463-5467, using exonuclease III-mediated unidirectionaldeletion (Ozkaynak et al., (1987) BioTechniques, 5:770-773), subcloningof restriction fragments, and synthetic primers. Compressions wereresolved by performing the reactions at 70° C. with Taq polymerase andrising 7-deaza-GTP (U.S. Biochemical Corp., Cleveland, Ohio).

As will be discussed in more detail below, the 1-3317 region of SEQ. IDNo. 1 contains a plurality of conserved DNA sequences which sharehomology with Pax homeobox consensus sequences of Epstein et al. (1994)J. Biol. Chem. 269:8355-8361, and have been identified and designatedherein as Pax responsive OP-1 modulating elements, comprisingapproximately nucleotides 108-121, 139-154, 157-167, 365-378, 491-503,497-511, 737-747, 891-903, 994-1006, 1123-1140, 1144-1161, 1285-1297,598-613, 1750-1762, 2001-2023, 2365-2378, 2931-2944 of SEQ. ID No. 1.Particularly preferred Pax 6 responsive OP-1 modulating elements resideat approximately nucleotides 108-121, 139-154, 365-378, 497-511,598-613, 1750-1762, 2365-2378 and 2931-2944. Other preferred Pax 6elements include approximately 157-167, 1123-1140, 1144-1161, 1285-1297and 2001-2023. Particularly preferred Pax 2 responsive OP-1 modulatingelements reside at approximately nucleotides 491-503, 737-747, 891-903and 994-1006.

Still other preferred Pax 6 responsive OP-1 modulating elements resideat approximately 104-120, 105-121, 142-158, 362-378, 497-513, 1750-1766,2362-2378, 2928-2944, 1128-1144, 1125-1141, 1143-1159, 2000-2016,2003-20019, 2007-2023, 601-617, 595-611, and 1746-1762. Still otherpreferred Pax 2 responsive OP-1 modulating elements reside atapproximately 493-503, 891-901 and 996-1006.

The transcription initiation site for the human OP-1 gene is at base2790 of SEQ. ID No. 1. The OP-1 protein translation initiation site isnucleotide 3318 of SEQ. ID No. 1.

As described above, osteogenic protein-1 plays a critical role inmodulating mesenchymal differentiation and inducing the process ofcartilage and bone formation. It is also required for kidney developmentand is shown to prevent kidney damage from ischemia/reperfusion injuryin rats. As presented below in Example 2, functional analysis of thehuman OP-1 gene promoter has been carried out. The OP-1 promoter and 5′truncated versions of it were placed upstream of the luciferase codingregion which served as the reporter gene. In certain embodiments, theresults of the luciferase assays after transient transfection of theseconstructs into a human kidney cell line, G401, indicate that thisupstream sequence has promoter activity. This activity resides about 300to 700 nucleotides upstream of the first ATG of the OP-1 gene. Thispromoter activity is constitutive and is not influenced bytransactivating agents, e.g., Pax 2 and Pax 6 (see FIG. 1B) which areinvolved in epithelial/mesenchymal interactions during tissuemorphogenesis. In other embodiments drawn to the region between 700 to3300 nucleotides upstream of the OP-1 gene, however, the data indicatethe presence of potential silencer elements of the promoter. Moreover,this region of the promoter can be influenced by a host of factors,including Pax 2, Pax 6 and Retinoic Acid, as illustrated in Example 2.

Both transient and permanent G401 cell lines containing thebelow-described constructs have been isolated and tested. Other hostcell lines including Human Retinoblastoma Y79, Rat Osteosarcoma ROS andPorcine Proximal Tubule LLCPK1 have been tested in transienttransfection assays. These data are expected to illustrate similarPax-mediated regulatory considerations and tissue specificity, therebypermitting even further identification of tissue- anddevelopmental-specific morphogen modulators.

EXAMPLE 2 Analysis of OP-1 Gene Expression

In one aspect, this invention presents a method in which OP-1 non-codingsequences are assayed while in operative association with a reportergene and modulator compounds such as Pax 2 or Pax 6 or analogs thereof,are thereby tested for their influence on the expression of OP-1. Forexample, non-coding sequences which are involved in the modulation ofOP-1 expression via Pax gene products or functional equivalents oranalogs thereof will be identified by (1) transfecting a cell with oneor more Pax protein expression vectors and an expression vectorcomprising OP-1 non-coding sequences in operative association with areporter gene; (2) culturing the transfectants with one or morecandidate compounds; (3) measuring the level of reporter geneexpression; and (4) comparing this level of expression to the level ofreporter gene expression in the absence of the compound(s).Alternatively, non-coding sequences can be assayed for theirresponsiveness to compounds which can mimic Pax 2 and/or Pax 6, i.e.,Pax 2 and/or Pax 6 analogs. The protocol of the present invention isbased on a procedure for identifying compounds which alter, eitherdirectly or indirectly, endogenous levels of morphogen expression.

It is also contemplated that candidate compounds may be administered invivo to modulate the level of an endogenous protein, such as OP-1. Thiscan be accomplished by detection of the expression product either at theprotein or RNA level.

Cultured cells are transfected with portions of OP-1 non-codingsequences in operative association with a reporter gene, and suchtransfected cells are maintained with the vector remaining as a plasmidin the cell nucleus, or the vector can be integrated into the host cellgenome, preferably at the OP-1 genomic locus. Cell samples for testingthe level of reporter gene expression are collected periodically andevaluated for reporter gene expression using the appropriate assay forthe given reporter gene as indicated in the section describing reportergene assays, or, alternatively, a portion of the cell culture itself canbe collected periodically and used to prepare polyA(+) RNA for mRNAanalysis. For example, to ascertain the particular time point at whichOP-1 is produced following treatment with a candidate morphogen, cellstreated with the candidate OP-1 modulating compound, cells treated withcompound are collected periodically and evaluated for OP-1 production,as described above. To monitor de novo OP-1 synthesis, some cultures arelabeled according to conventional procedures with an³⁵S-methionine/³⁵S-cysteine mixture for 6-24 hours and then evaluated toquantitate OP-1 synthesis by conventional immunoassay methods.Alternatively, anti-OP-1 antibodies can be labeled and incubated withthe cells or cell lysates, and the bound complexes detected andquantitated by conventional means, such as those described herein above.Tissues can also be examined directly for the synthesis of OP-1 mRNAusing the art-recognized technique of in situ hybridization.

Once candidate compounds are identified, they can be produced inreasonable, useful quantities using standard methodologies known in theart. Amino acid-based molecules can be encoded by synthetic nucleic acidmolecules, and expressed in a recombinant expression system as describedherein above or in the art. Alternatively, such molecules can bechemically synthesized, e.g., by means of an automated peptidesynthesizer, for example. Non-amino acid-based molecules can be producedby standard organic chemical synthesis procedures.

Morphogen agonists are anticipated to have utility in any applicationwhere tissue morphogenesis is desired, such as in the regeneration ofdamaged tissue resulting from mechanical or chemical trauma,degenerative diseases, tissue destruction resulting from chronicinflammation, cirrhosis, inflammatory diseases, cancer and the like, andin the regeneration of tissues, organs and limbs. Morphogen antagonistsare envisioned to have utility in applications where tissuemorphogenesis is to be limited as, for example, in the treatment ofmalignant transformations including, but not limited to, osteosarcomas,Paget's disease, and fibrodysplasia ossificans progressiva (See, forexample, Roush (1996) Science 273:1170). The ability to detect OP-1protein in solution provides a valuable tool for diagnostic assays,allowing us to monitor the levels of OP-1 free in the body, e.g., inserum, urine, spinal or peritoneal fluid, breast exudate, and other bodyfluids in order to assess the presence of OP-1 modulating activity. Thepresent invention therefor provides a means of identifying naturallyoccurring or synthetic proteins or other factors or drugs that have OP-1modulating activities. Moreover, compounds that induce the expression ofPax 2 or Pax 6 are also particularly useful.

OP-1 is expressed in a variety of different cell types, including renal,bone, lung, heart, uterine, cardiac and neural tissue. Candidatecompounds can be identified which have a modulating effect on cells ofone tissue type but not another, and/or wherein the effect is modulateddifferently in the different cells. The assay described below can beused to evaluate the effect of a candidate compound in a particular celltype known to express OP-1 under physiological conditions.

The present invention therefor also provides a test cell or cell linewhich expresses an exogenously introduced OP-1 protein, which isresponsive to morphogen modulators such as Pax 2 and/or Pax 6 or analogsthereof as contemplated herein. Said cell or cell line can be also usedas described herein, to replace cell lines that produce OP-1endogenously, for the screening assays described herein above. Further,the invention provides a test cell that is transfected with DNA encodingPax gene products (e.g., Pax 2 and/or Pax 6) for the modulation ofendogenous or exogenous OP-1 gene expression.

While a readily assayable, well characterized, non OP-1 reporter gene ispreferred in the screening method disclosed herein, as will beappreciated by those having ordinary skill in the art, OP-1 codingsequence also may be used in the method of the invention providing animproved method for the production of high levels of OP-1, exploitingthe high level of induction of gene expression of the present invention.The OP-1 expression preferably is determined by an immunoassay or byNorthern or dot blot or other means for measuring mRNA transcript. See,for example, WO 95/11983, published May 4, 1995 for a detaileddescription on assaying changes in OP-1 levels in a cell or fluid.Further, the cells of the present invention can be used in the analysisof the transcription factors, for example, DNA-protein and/orprotein-protein interactions involved in OP-1 transcription regulationor that of other morphogens.

Provided below is an exemplary protocol for carrying out the method ofthe invention, using the luciferase gene as the reporter gene and amammalian cell line known to express OP-1. The example is non-limiting,and other cells, reporter genes and morphogen non-coding sequences suchas, but not limited to, an OP-1 non-coding sequence, are envisioned.

The OP-1 genomic DNA nucleotides 1-3317 of SEQ. ID No. 1 was used toprepare a series of deletion constructs carrying the luciferase reportergene and portions of the OP-1 gene non-coding region (FIG. 1A). ThepGL2-Basic plasmid comprising a nucleotide sequence encoding thedetectable enzyme luciferase (Promega, Madison Wis.) was employed as thebasic vector. The OP-1 promoter sequence (corresponding to nucleotides 1to 3305 of SEQ. ID No. 1) was then inserted into the pGL2-Basic plasmid(construct pAS3.3, FIG. 1A). Nucleotide +1 is 30 nucleotides downstreamof the Hind III site present in the OP-1 genomic sequence. There aretherefore an additional 30 base pairs of DNA sequence upstream ofposition +1 in the OP-1 promotor, comprising AAGCTTGATG CCTGCACAGTCAGCCCTCAG (SEQ ID NO: 10), wherein AAGCTT corresponds to a Hind IIIsite.

The genomic sequence was digested with Hind III and BamHI. The BamHIsite was introduced into the OP-1 promoter at nucleotides 3306 to 3311of SEQ. ID No. 1 by site-directed mutagenesis according to methods wellknown in the art using the following reverse complement primer: 5′ CATCGC GCC GGA TCC ACG CGC TAC CCG GGC 3′ (SEQ ID NO: 11) wherein GGATCCcorresponds to a BamHI site.

Thus the mutagenesis generated the following change in the OP-1sequence:

native seq: 5′ GCC CGG GTA GCG CGT AGA GCC GGC GCG ATG 3′ (SEQ ID NO:12)

altered seq: 5′ GCC CGG GTA GCG CGT GGA TCC GGC GCG ATG 3′ (SEQ ID NO:13)

This resulted in the following changes in the OP-1 promotor in constructpAS3.3: GCCCGGGTAG CGCGTGGATC TAAGTAAGCT TGGCATTCCG GTACTGTTGG TAAAATG(SEQ ID NO: 14) wherein the G represents nucleotide 3306 of SEQ. ID No.1 and wherein ATG corresponds to the luciferase ATG (nucleotides 3318 to3320 of SEQ. ID No. 1).

Therefore, the native OP-1 promoter sequences stop at nucleotide 3305,nucleotide 3306 results from an A to G nucleotide change, andnucleotides 3307 to 3345 are derived from the pGL2-Basic vector. Thus,3335 nucleotides (30 nucleotides upstream of nucleotide 1 up to 3305 ofSEQ. ID No. 1) of cloned 5′ non-coding sequence of the OP-1 gene wasplaced in transcriptionally operative association with the luciferasereporter gene.

Serial 5′ deletions (FIG. 1A) were constructed by digestion of thepAS3.3 construct with a series of restriction enzymes followed byreligation of the plasmid according to well-known cloning techniques.For example, the pAS3.3 vector was cleaved with BglII (present in thepGL2-Basic vector and in the OP-1 promoter) and recircularized to obtainpAS2.5; the pAS3.3 vector was cleaved with KpnI (present in thepGL2-Basic vector and in the OP-1 promoter) and recircularized to obtainpAS1.2; the pAS3.3 vector was cleaved with XhoI (present in thepGL2-Basic vector) and SacII, followed by a T4 DNA polymerase fill-inreaction and recircularized to obtain pAS0.8; the pAS3.3 vector wascleaved with KpnI (present in the pGL2-Basic vector) and AatII followedby a T4 DNA polymerase fill-in reaction and recircularized to obtainpAS0.7; the pAS3.3 vector was cleaved with XhoI (present in thepGL2-Basic vector) and PvuII followed by a T4 DNA polymerase fill-inreaction and recircularized to obtain pAS0.6; the pAS3.3 vector wascleaved with XhoI (present in the pGL2-Basic vector) and AvrII followedby a T4 DNA polymerase fill-in reaction and recircularized to obtainpAS0.3; and the pAS3.3 vector was cleaved with KpnI (present in thepGL2-Basic vector) and PstI followed by a T4 DNA polymerase fill-inreaction and recircularized to obtain pAS0.1.

The vectors were transfected into G401 human kidney cells, either aloneor together with the cDNA for human Pax 2 and/or for human Pax 6,previously cloned into a pCMVβ expression vector (Clonetech), with theremoval of the β-galactosidase gene by standard methods (Epstein, et al.(1994) J. Biol. Chem. 269:8355-8361), the disclosure of which isincorporated herein by reference. Briefly, cells were plated in 60 mmpetri dishes (5×10⁵ cell/dish) in McCoy's 5A medium containing 10% fetalbovine serum. Twenty-four hours later, the above-described vectors weretransfected into the cultured cells, using a Lipofectamine (BRL/Gibco)method in serum-free medium (Optimem, BRL) for 6 hours. Cells weretransfected with (a) an OP-1 promoter constructs alone; (b) an OP-1promoter construct+the Pax 2 construct; (c) an OP-1 promoterconstruct+the Pax 6 construct; or (d) an OP-1 promoter construct+the Pax2 construct+the Pax 6 construct. Control transfected cells contained thepGL-Basic plasmid without OP-1 non-coding sequences as a negativecontrol and pSV-Luc, which has an SV40 promoter, as a positive control.Thereafter, transfected cells were cultured in complete mediaSeventy-two hours later, luciferase activity was measured using thePromega Luciferase Assay System (Promega, Madison Wis.) and aluminometer (DynaTech). The results are displayed in FIG. 1B anddiscussed below.

The luciferase production by cells containing the pAS3.3 construct,comprising 3305 nucleotides upstream of the translation start site ofthe OP-1 gene, was induced 2.4 fold by co-transfection with the Pax 2cDNA and 9.6 fold by co-transfection with the Pax 6 cDNA (FIG. 1B). Thetriple transfection of both the Pax 2 and Pax 6 expression vectors withthe pAS3.3 construct resulted in an additive 11.5 fold inductionsuggesting that Pax 2 and Pax 6 gene products have an additive effect onOP-1 gene expression. Cells containing the 5′ deletion mutants of thepAS3.3 construct were less responsive to the inductive effect ofco-transfection with Pax 2 or Pax 6 constructs. For example, the pAS2.5construct was only induced 1.7 fold by co-transfection with the Pax 2construct and this Pax 2 responsiveness was abolished by furtherdeletion of about 1.3 kb of 5′ sequence, with no Pax 2 inductiondetected for the pAS1.2 construct or the remaining constructs withfurther 5′ deletions.

Pax 6 responsiveness was also lost by about 42% upon deletion of 793bases from the pAS3.3 construct A 4 to 5.5 fold induction of luciferaseproduction was demonstrated in the cells comprising the Pax 6 constructco-transfected with pAS2.5 (+794 to +3305), pAS1.2 (+2068 to +3305),pAS0.8 (+2501 to +3305), and pAS0.7 (+2606 to +3305) of SEQ. ID No. 1but this Pax 6 responsiveness was lost upon deletion of nucleotides+2606 to +2690. This suggests that this 85 nucleotide region containspreferred sequences necessary for OP-1 modulation by Pax gene productsor analogs thereof.

Constructs pAS3.3 and pAS2.5, comprising 3.3 kb 5′ non-coding region (1to 3305 of SEQ. ID No. 1) and a 793 nucleotide 5′ deletion thereof,respectively, produced less luciferase constitutively than the otherdeletion mutants, suggesting the presence of negative regulatoryelements in the 5′ non-coding region comprising nucleotides 1 to 2067(FIG. 1B).

The complete DNA sequence of the 5′ noncoding upstream OP-1 gene (1 to3317 of SEQ. ID No. 1) was analyzed using a string search program toidentify putative Pax 2 or Pax 6 binding motifs, relying in part on thePax 2 and Pax 6 consensus sequences described by Epstein et al. (1994)J. Biol. Chem. 269:8355-8361, and in part on certain other suitable Paxconsensus sequences disclosed herein. One consensus sequence for Pax 6is T T C A C G C A/T T G/C A N T G/T A/C N T/C corresponding to SEQ. IDNo. 2 wherein the N is either A, G, C or T. One consensus sequence forPax 2 is G T C A C/T G C G/A T G A and corresponds to SEQ. ID No. 3. Thestring search program allowed for partial mismatches and revealed anumber of Pax 6 or Pax 2 responsive regions. Currently preferred Pax 2responsive regions are located at approximately 491-503, 737-747,891-903, 994-1006 of SEQ. ID No. 1 and share approximately 69-82%identity with the Pax 2 consensus sequence of SEQ. ID. No. 3.

Currently preferred Pax 6 responsive elements include approximatelynucleotides 108-121, 139-154, 365-378, 497-511, 1750-1762, 2365-2378,2931-2944 of SEQ. ID No. 1. These Pax 6 responsive elements sharebetween 53% and 82% identity with the Pax 6 consensus sequence of SEQ.ID No. 3. Other currently preferred Pax 6 responsive elements includeapproximately nucleotides 157-167, 1123-1140, 1144-1161, 1285-1297, and2001-2023 of SEQ. ID No. 1.

It is understood that some of the consensus Pax motifs may function toregulate OP-1 gene expression, where others may not. Further,characterization of each putative Pax responsive OP-1 element isaccomplished as described below in Example 5. As earlier discussed, itis contemplated that Pax responsive elements, such as those describedherein, are present in the promoter sequences of other morphogens asdefined herein and thereby can be identified and exploited as taughtherein. Pax responsive elements, as contemplated by the presentinvention, modulate the in vivo, ex vivo and in vitro expression ofmorphogens and thus are useful for identifying particular compounds thatcan modulate particular morphogens.

It is contemplated that the present invention further provides a usefulmethod for identifying nucleic acids and their respective encodedcompounds capable of modulating Pax mediated OP-1 gene regulation. Inone embodiment, one or more candidate nucleic acids which encode acandidate compound or compounds capable of intracellular modulation ofOP-1 gene expression, mediated directly or indirectly by Pax responsiveelements located in the OP-1 gene 5′ non-coding region (nucleotides1-3317 of SEQ. ID No. 1), are also transfected into the cells of theinvention comprising Pax responsive OP-1 5′ non-coding sequence/reportergene vectors. “Candidate nucleic acid” or “candidate DNA” or “candidateRNA” as defined herein, is therefor a nucleic acid (e.g. a DNA or RNA)that encodes a compound that modulates OP-1 gene expression via a Paxresponsive element. In one embodiment, a candidate DNA can encode atranscription factor that shares functional similarity with a Pax geneproduct or is a Pax analog or agonist. Alternatively, the candidatenucleic acid can encode a compound that behaves as an inhibitor of OP-1gene expression or is a Pax antagonist. In another embodiment, acandidate RNA or RNAs may be first isolated from a cell or synthesizedin vitro by methods well known to the skilled artisan and then used totransfect the above-described cell. It is also, contemplated that tissuespecific candidate nucleic acids can be identified and isolatedaccording to the method of the present invention. Thus, for example,expression vectors comprising tissue specific candidate DNAs can beco-transfected into a cell together with an expression vector containingthe OP-1 5′ non-coding sequence of nucleotides 1-3317 of SEQ ID No 1, ortruncated versions thereof, in operative association with a luciferasereporter gene. Alterations in luciferase activity relative to controltransfections is an indication that the candidate DNA encodes a compoundcapable of modulating OP-1 gene regulation. It is contemplated thatindividual nucleic acids or pools of nucleic acids, for example, a cDNAlibrary or a pool of synthetic RNAs, may be screened for their abilityto modulate OP-1 gene expression via the responsive elements of thepresent invention in accordance with the methods of the invention.

EXAMPLE 3 Further Characterization of the Pax Responsive OP-1 ModulatingElements

To characterize the respective roles of certain of the Pax 2 (forexample, approximately, nucleotides 491-503, 737-747, 891-903, 994-1006of SEQ. ID No. 1) and Pax 6 responsive elements (for example,approximately, nucleotides 108-121, 139-154, 157-167, 365-378, 498-511,598-613, 1123-1140, 1144-1161, 1285-1297, 1750-1762, 2001-2023,2365-2378, 2931-2944 of SEQ. ID No. 1) in the modulation of the OP-1gene expression, mutations of the various Pax 2 and Pax 6 responsiveelements are analyzed. A loss or decrease of Pax mediated OP-1 geneexpression by mutation of a portion of each Pax 2 responsive elementsequence indicates a role for that sequence in Pax mediated OP-1 generegulation. Conversely, an increase in Pax mediated OP-1 gene expressionof the mutated Pax responsive elements may indicate, for example,enhanced binding of Pax transcription factors to the altered DNArecognition sequence or, alternatively, a loss or decrease in binding ofone or more negative regulatory elements.

Further, the OP-1 gene promoter is analyzed by DNase footprinting tofurther define the Pax responsive regions and to determine bindingspecificity and affinity of the Pax gene products required for theexpression of the OP-1 gene. DNase footprinting is carried out accordingto techniques well known to those skilled in the art.

In addition, the interaction of the OP-1 promoter with nuclear proteinsis further characterized by gel shift assays using oligonucleotidesprobes corresponding to the Pax 2 consensus sequence (SEQ. ID No. 3) orthe Pax 6 consensus sequence (SEQ. ID No. 2) or variants thereof, withand without mutations in the oligonucleotide sequences. Briefly, cellsare lysed according to art known methods and nuclear extracts areprepared and incubated with a radiolabelled oligonucleotide of choice,by methods well known to the skilled artisan. Binding of one or more ofthe protein components of the extract to the oligonucleotide producesDNA/protein complexes having retarded electrophoretic mobility relativeto the mobility of the uncomplexed oligonucleotide DNA probe. Completionof radiolabelled oligonucleotide binding with cold (unlabelled)oligonucleotide is performed to confirm the specificity of DNA/proteincomplexes. These assays can demonstrate alterations in the amount oractivity of a nuclear extract component in response to treatment withcandidate compounds.

Supershift assays, in which the nuclear extract oligonucleotidecomplexes are further retarded by complexing with specific antibody, areused to determine the identity of the Pax gene product or analogthereof. In these assays, the previously describedprotein-oligonucleotide probe complexes are farther incubated withspecific antibody and subjected to electrophoresis, according to artknown methods. The DNA-protein complexes from a test cell induced, forexample, by a candidate compound, are supershifted during gel analysiswhen treated with an anti-Pax 2 or anti-Pax 6 antibody, or other Paxfamily antibodies, for example. This supershift can be reversed byincubation with the antigenic peptide.

The tissue specificity of the Pax-DNA complexes is further defined by acomparison of gel shift and supershift mobilities using cell extractsfrom various tissues and cells. These studies may reveal the involvementof one or more tissue specific factors that may participate in Paxmediated regulation of OP-1 gene transcription.

EXAMPLE 4 In vivo Animal Model for Testing Efficacy of Compounds toModulate OP1 Expression

It previously has been demonstrated that OP-1 can be an effectivetreatment for osteoporosis on the standard ovariectomized rat model, asindicated by the dose-response increase in alkaline phosphatase andosteocalcin levels following injection with OP-1. The osteoporotic ratmodel provides an in vivo model for evaluating the efficacy of acandidate compound for modulating morphogen synthesis. In order todetermine the effect of a candidate morphogen stimulating agent on OP-1production and, thereby, on bone production in viva, alkalinephosphatase and osteocalcin levels are measured under conditions whichpromote osteoporosis, e.g., wherein osteoporosis is induced by ovaryremoval in rats and in the presence and absence of a candidate compound.A compound competent to enhance or induce endogenous OP-1 expressionshould result in increased osteocalcin and alkaline phosphatase levels.

Forty Long-Evans rats (Charles River Laboratories, Wilmington) weighingabout 200 g each are ovariectomized (OVX) using standard surgicalprocedures, and ten rats are sham operated. The ovariectomization of therats produces an osteoporotic condition within the rats as a result ofdecreased estrogen production. Food and water are provided ad libitum.Eight days after ovariectomy, the rats, prepared as described above, aredivided into three groups: (a) sham-operated rats; (b) ovariectomizedrats receiving 1 ml of phosphate-buffered saline (PBS) i.v. in the tailvein; and (c) ovariectomized rats receiving various dose ranges of thecandidate morphogen stimulating agent either by intravenous injectionthrough the tail vein or direct administration to kidney tissue.

The effect of the candidate compound on in vivo bone formation can bedetermined by preparing sections of bone tissue from the ovariectomizedrats. Each rat is injected with 5 mg of tetracycline, which will stainthe new bone (visualized as a yellow color by fluorescence), on the 15thand 21st day of the study, and on day 22 the rats are sacrificed. Thebody weights, uterine weights, serum alkaline phosphatase levels, serumcalcium levels and serum osteocalcin levels then were determined foreach rat. Bone sections are prepared and the distance separating eachtetracycline staining is measured to determine the amount of new bonegrowth. The levels of OP-1 in serum following injection of the candidateagent also can be monitored on a periodic basis using routine methods.

EXAMPLE 5 Determination of OP-1 Protein Production

Where OP-1 acts as the reporter gene, detection of the gene productreadily can be assayed using antibodies specific to the protein andstandard immunoassay testings. For example, OP-1 may be detected using apolyclonal antibody specific for OP-1 in an ELISA, as follows.

1 μg/100 μl of affinity-purified polyclonal rabbit IgG specific for OP-1is added to each well of a 96-well plate and incubated at 37° C. for anhour. The wells are washed four times with 0.167M sodium borate bufferwith 0.15 M NaCl (BSB), pH 8.2, containing 0.1% Tween 20. To minimizenon-specific binding, the wells are blocked by filling completely with1% bovine serum albumin (BSA) in BSB and incubating for 1 hour at 37° C.The wells are then washed four times with BSB containing 0.1% Tween 20.A 100 μl aliquot of an appropriate dilution of each of the test samplesof cell culture supernatant is added to each well in triplicate andincubated at 37° C. for 30 min. After incubation, 100 ll biotinylatedrabbit anti-OP-1 serum (stock solution is about 1 mg/ml and diluted1:400 in BSB containing 1% BSA before use) is added to each well andincubated at 37° C. for 30 min. The wells are then washed four timeswith BSB containing 0.1% Tween 20. 100 μl streptavidin-alkaline(Southern Biotechnology Associates, Inc. Birmingham, Ala., diluted1:2000 in BSB containing 0.1% Tween 20 before use) is added to each welland incubated at 37° C. for 30 min. The plates are washed four timeswith 0.5M Tris buffered Saline (TBS), pH 7.2. 50 μl substrate (ELISAAmplification System Kit, Life Technologies, Inc., Bethesda, Md.) isadded to each well incubated at room temperature for 15 min. Then, 50 μlamplifier (from the same amplification system kit) is added andincubated for another 15 min. at room temperature. The reaction isstopped by the addition of 50 μl 0.3 M sulfuric acid. The OD at 490 nmof the solution in each well is recorded. To quantitate OP-1 in culturemedia, a OP-1 standard curve is performed in parallel with the testsamples.

EXAMPLE 6 Production of OP-1 Polyclonal and Monoclonal Antibodies

Polyclonal antibody for OP-1 protein may be prepared as follows. Eachrabbit is given a primary immunization of 100 μg/500 μl E. coli producedOP-1 monomer (amino acids 328-431 in SEQ ID No:5) in 0.1% SDS mixed with500 μl Complete Freund's Adjuvant. The antigen is injectedsubcutaneously at multiple sites on the back and flanks of the animal.The rabbit is boosted after a month in the same manner using incompleteFreund's Adjuvant. Test bleeds are taken from the ear vein seven dayslater. Two additional boosts and test bleeds are performed at monthlyintervals until antibody against OP-1 is detected in the serum using anELISA assay. Then, the rabbit is boosted monthly with 100 μg of antigenand bled (15 ml per bleed) at days seven and ten after boosting.

Monoclonal antibody specific for OP-1 protein may be prepared asfollows. A mouse is given two injections of E. coli produced OP-1monomer. The first injection contains 100 μg of OP-1 in completeFreund's adjuvant and is given subcutaneously. The second injectioncontains 50 μg of OP-1 in incomplete adjuvant and is givenintraperitoneally. The mouse then receives a total of 230 μg of OP-1 infour intraperitoneal injections at various times over an eight monthperiod. One week prior to fusion, both mice are boostedintraperitoneally with 100 μg of OP-1 and 30 μg of the N-terminalpeptide (Ser₂₉₃-Asn₃₀₉-Cys) conjugated through the added cysteine tobovine serum albumin with SMCC crosslinking agent. This boost wasrepeated five days (IP), four days (IP), three days (IP) and one day(IV) prior to fusion. The mouse spleen cells are then fused to myelomacells at a ratio of 1:1 using PEG 1500 (Boeringer Mannheim), and thecell fusion is plated and screened for OP-1-specific antibodies usingOP-1 as antigen. The cell fusion and monoclonal screening are thenperformed according to standard procedures widely available in the art.

EXAMPLE 7 Process for Detecting OP-1 in Serum

Presented below is a sample protocol for identifying OP-1 in serum.Following this general methodology, OP-1 may be detected in body fluids,including serum, and can be used in a protocol for evaluating theefficacy of an OP-1 modulating compound in vivo.

A monoclonal antibody raised against mammalian, recombinantly producedOP-1 using standard immunology techniques well described in the art anddescribed generally in example 6, above, was immobilized by passing theantibody over an agarose-activated gel (e.g., Affi-Gel™, from Bio-RadLaboratories, Richmond, Calif., prepared following manufacturer'sinstructions) and used to purify OP-1 from serum. Human serum then waspassed over the column and eluted with 3M K-thiocyanate. K-thiocyanatefractions then were dialyzed in 6M urea, 20 mM PO₄, pH 7.0, applied to aC8 HPLC column, and eluted with a 20 minute, 25-50% acetonitrile/0.1%TFA gradient Mature, recombinantly produced OP-1 homodimers elutebetween 20-22 minutes, and are used as a positive control. Fractionsthen were collected and tested for the presence of OP-1 by standardimmunoblot using an OP-1 specific antibody. Using this method OP-1readily was detected in human serum. See also, PCT/US92/07432 for adetailed description of the assay.

EXAMPLE 8 Considerations for Formulation and Methods for AdministeringTherapeutic Agents

Where the OP-1-modulating agent identified herein comprises part of atissue or organ preservation solution, any commercially availablepreservation solution may be used. For example, useful solutions knownin the art include Collins solution, Wisconsin solution, Belzersolution, Eurocollins solution and lactated Ringer's solution.Generally, an organ preservation solution usually possesses one or moreof the following properties: (a) an osmotic pressure substantially equalto that of the inside of a mammalian cell, (solutions typically arehyperosmolar and have K+ and/or Mg++ ions present in an amountsufficient to produce an osmotic pressure slightly higher than theinside of a mammalian cell); (b) the solution typically is capable ofmaintaining substantially normal ATP levels in the cells; and (c) thesolution usually allows optimum maintenance of glucose metabolism in thecells. Organ preservation solutions also may contain anticoagulants,energy sources such as glucose, fructose and other sugars, metabolites,heavy metal chelators, glycerol and other materials of high viscosity toenhance survival at low temperatures, free oxygen radical inhibitingagents and a pH indicator. A detailed description of preservationsolutions and useful components may be found, for example, in U.S. Pat.No. 5,002,965.

Where the OP-1-modulating agent is to be provided to an individual,e.g., the donor prior to harvest, or the recipient prior to orconcomitant with transplantation, the therapeutic agent may be providedby any suitable means, preferably directly (e.g., locally, as byinjection to the tissue or organ locus) or systemically (e.g.,parenterally or orally).

Useful solutions for parenteral administration may be prepared by any ofthe methods well known in the pharmaceutical art, described, forexample, in Remington's Pharmaceutical Sciences (Gennaro, A., ed.), MackPub., 1990. Formulations may include, for example, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable original, hydrogenatednaphthalenes, and the like. Formulations for direct administration, inparticular, may include glycerol and other compositions of highviscosity to help maintain the agent at the desired locus.Biocompatible, preferably bioresorbable, polymers, including, forexample, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate,lactide and glycolide polymers and lactide/glycolide copolymers, may beuseful excipients to control the release of the agent in vivo.

As will be appreciated by those skilled in the art, the concentration ofthe compounds described in a therapeutic composition will vary dependingupon a number of factors, including the dosage of the drug to beadministered, the chemical characteristics (e.g., hydrophobicity) of thecompounds employed, and the route of administration. Where themorphogen-stimulating agent is part of a preservation solution, thedosage likely will depend for example, on the size of the tissue ororgan to be transplanted, the overall health status of the organ ortissue itself, the length of time between harvest and transplantation(e.g., the duration in storage), the frequency with which thepreservation solution is changed, and the type of storage anticipated,e.g., low temperature. In general terms, preferred ranges include aconcentration range between about 0.1 ng to 100 μg/kg per tissue ororgan weight per day.

Where the therapeutic agent is to be administered to a donor orrecipient, the preferred dosage of drug to be administered also islikely to depend on such variables as the type and extent of progressionof the disease, the overall health status of the particular patient, therelative biological efficacy of the compound selected, the formulationof the compound excipients, and its route of administration. In generalterms, a suitable compound of this invention may be provided in anaqueous physiological buffer solution containing about 0.001% to 10% w/vcompound for parenteral administration. Typical dose ranges are fromabout 10 ng/kg to about 1 g/kg of body weight per day; and preferreddose range is from about 0.1 μg/kg to 100 mg/kg of body weight per day.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

EXAMPLE 9 An AatII-PvuII Fragment of the OP-1 Upstream Region Containsan Expression Silencer

Luciferase levels were assayed in G401 cells transfected with the OP-1upstream deletion constructs described in Example 2. The G401 cells weretransfected with the deletion constructs using Lipofectamine.Forty-eight to seventy-two hours later, luciferase activity was measuredusing the Promega Luciferase Assay System (Promega, Madison Wis.) and aluminometer (DynaTech). The relative luciferase activities are shown inFIG. 2. Maximal activity was observed for the construct containing 0.6kb of OP-1 upstream DNA. The addition of approximately 0.1 kb ofupstream DNA (the AatII-PvuII fragment, approximately nucleotides2606-2690 of SEQ ID NO: 1) reduces luciferase activity approximatelyfive fold as shown in FIG. 2 by the AatII construct containingapproximately 0.7 kb of OP-1 upstream DNA.

The expression silencing property of the AatII-PvuII fragment(nucleotides 2606-2690 of SEQ ID NO: 1) was further tested by comparingluciferase expression in pAS33 containing cells with luciferaseexpression in pAS3.3d containing cells. The pAS3.3 construct contains3.3 kb of OP-1 upstream DNA fused to the luciferase gene. The pAS3.3dconstruct lacks the AatII-PvuII fragment, and was made by digesting thepAS3.3 construct with AatII and PvuII, blunting the AatII overhang, andreligating the vector.

G401 cells were plated in triplicate in 6 well plates. After a one dayincubation, the cultured cells were transfected with the deletionconstructs, using Lipofectamine and approximately 1 μg of either pAS3.3or pAS3.3d. Forty-eight to seventy-two hours later, luciferase activitywas measured as described previously. The results are shown in FIG. 3.Cells containing pAS3.3 expressed approximately three-fold (between twoand four-fold) more luciferase than cells containing pAS3.3d. TheAatII-PvuII fragment (nucleotides 2606-2690 of SEQ ID NO: 1) thereforecontains a silencer of gene expression.

The luciferase production by cells containing the pAS3.3 construct,comprising 3305 nucleotides upstream of the translation start site ofthe OP-1 gene, was induced 2.4 fold by co-transfection with the Pax 2cDNA and 9.6 fold by co-transfection with the Pax 6 cDNA (FIG. 1B). Thetriple transfection of both the Pax 2 and Pax 6 expression vectors withthe pAS3.3 construct resulted in an additive 11.5 fold inductionsuggesting that Pax 2 and Pax 6 gene products have an additive effect onOP-1 gene expression. Cells containing the 5′ deletion mutants of thepAS3.3 construct were less responsive to the inductive effect ofco-transfection with Pax 2 or Pax 6 constructs. For example, the pAS2.5construct was only induced 1.7 fold by co-transfection with the Pax 2construct and this Pax 2 responsiveness was abolished by furtherdeletion of about 1.3 kb of 5′ sequence, with no Pax 2 inductiondetected for the pAS1.2 construct or the remaining constructs withfurther 5′ deletions.

15 1 17415 DNA Homo sapiens 1 tcaaccggtc tctttaggtt ttggctgtgcttattactat tcattcaaca ggtactaatt 60 gagcacctgc tgtgtgccag gctcagaataggctcaggtg agatgcacaa agaagggtaa 120 actagaatcc ttgcttagac actgacggatcagttgtttc atatgtaaat tgtagcacca 180 agacctgctg cccctgcccc cagcctcacctgcttgtgaa gatccctcca aaagatttga 240 gagtagataa aaagcagaga ctactactgaagaacagggc tgctttggct ccttattatt 300 tcagactttg gaagaaaatg acctcctttttctctactgg cactgagtgc atagctgacc 360 tagcaagcca ggcctggagg gcgtgtgcagggctggggac cgagcctggt ttctgttccc 420 tgctctgcag ctcaagcact tgctgttcctccacctggga tgcctttccc tggaaaagcc 480 tgtctctttc ttgtctttca ggactcaggtcagtggcatc tcctccaaaa actccccttc 540 ccaccctcca tcacctcacc ctgtttatctgcgcccccgc ccccactgcc tgtcacttat 600 tgcaggctga agtgacccag gctctccagttgtacactct cagatggacc ctggacgact 660 gtggcactcc tgcaatttcc ccagtctccctggggtagga ttcctgcttg ccaggatgcc 720 cacctttcct tctccctcct gcatgtcctcctctgcctgg cttctgaatt gtttccagag 780 agagtgatag acaagatctg cctctccttcagtccctgaa tcttatttaa ggctcttgct 840 ttgcttccct ggcctggagg cggctccttgatggagtctg ccatgtgggt tcgctcatgg 900 ccatgtcttc ctgcccagca tggtgcttggccctgggact ggccacataa tatctgggcc 960 aggtgcaaaa ttagtacggg gcagggggtactttgttcat aggtgattca gaaccacata 1020 tggtgacctc agagtaggaa accaagtgtggggcccttaa gagctggggg gccctgtacg 1080 actgtccagg ttgcaggccc cacagctcgcctcctgatat cctgtgctcc atgcttgtct 1140 gttgaaggaa ggagtgaatg gatgaagagcaggtggtggg gggtggtttg agggccttgc 1200 tggtgggtgg gtagaggccc ctccctggcatggggctcaa gacctgttcc atcccacagc 1260 ctggggctgt gtgtaaatgg ccaggacctgcaggctggca tttttctgct ccttgcctgg 1320 ctctggctcc cctttctcca cccatgtggcccctcaggct gccatctagt ccaaaagtcc 1380 caagggagac ccagaggcca cttggcaaactacttctgct ccagaaaact gtagaagacc 1440 ataattctct tccccagctc tcctgctccaggaaggacag ccccaaagtg aggcttagca 1500 gagcccctcc cagacaagcg cccccgcttccccaacctca gcccttccca gttcatccca 1560 aaggccctct ggggacccac tctctcacccagccccagga gggaaggaga caggatgaac 1620 ttttaccccg ctgccctcac tgccactctgggtgcagtaa ttcccttgag atcccacacc 1680 ggcagaggga ccggtgggtt ctgagtggtctggggactcc ctgtgacagc gtgcatggct 1740 cggtattgat tgagggatga atggatgaggagagacagga gaggaggccg atggggaggt 1800 ctcaggcaca gacccttgga ggggaagaggatgtgaagac cagcggctgg ctccccaggc 1860 actgccacga ggagggctga tgggaagccctagtggtggg gctggggtgt ctggtctcag 1920 gctgaggggt ggctggaaag atacagggccccgaagagga ggaggtggga agaacccccc 1980 cagctcacac gcagttcact tattcactcaacaaatcgtg actgcgcacg tacagtggct 2040 accaggcgct gggttcaagg cactgcgggtaccagaggtg cggagaagat cgctgatccg 2100 ggccccagtg ctctgggtgt ctagcgggggtaagaaggca ataaagaagg cacggagtaa 2160 ctcaaacagc aattccagac agcaagagaaactacaggaa agaaaacaaa cgtgcgaggg 2220 gcgaggcgag gaaacaacct cagcttggcaggtcttggag gtctctggga ggagaaagca 2280 gcgtctgatg ggggcgggag gtggtgagtggggagaggtc caggcggagg gaatggcgag 2340 cgagagacag gctggcaacg gcttcagggaggcgcggagg ggtcagcgtg gctggcttaa 2400 aaggatacat gggactaggg gcaagaccggctcaaggtca ccgcttccag gaccttctat 2460 ttccgcgcca cctccgcgct cccccaacttttcccaccgc ggtccgcagc ccacccgtcc 2520 tgctcgggcc gccttcctgg tccggaccgcgagtgccgag agggcagggc cggctccgat 2580 tcctccagcc gcatccccgc gacgtcccgccaggctctag gcaccccgtg ggcactcagt 2640 aaacatttgt cgagcgctct agagggaatgaatgaaccca ctgggcacag ctggggggag 2700 ggcggggccg agggcaggtg ggaggccgccggcgcgggag gggcccctcg aagcccgtcc 2760 tcctcctcct cctcctccgc ccaggccccagcgcgtacca ctctggcgct cccgaggcgg 2820 cctcttgtgc gatccagggc gcacaaggctgggagagcgc cccggggccc ctgctatccg 2880 cgccggaggt tggaagaggg tgggttgccgccgcccgagg gcgagagcgc cagaggagcg 2940 ggaagaagga gcgctcgccc gcccgcctgcctcctcgctg cctccccggc gttggctctc 3000 tggactccta ggcttgctgg ctgctcctcccacccgcgcc cgcctcctca ctcgcctttt 3060 cgttcgccgg ggctgctttc caagccctgcggtgcgcccg ggcgagtgcg gggcgagggg 3120 cccggggcca gcaccgagca gggggcgggggtccgggcag agcgcggccg gccggggagg 3180 ggccatgtct ggcgcgggcg cagcggggcccgtctgcagc aagtgaccga gcggcgcgac 3240 ggccgcctgc cccctctgcc acctggggcggtgcgggccc ggagcccgga gcccgggtag 3300 cgcgtagagc cggcgcgatg cacgtgcgctcactgcgagc tgcggcgccg cacagcttcg 3360 tggcgctctg ggcacccctg ttcctgctgcgctccgccct ggccgacttc agcctggaca 3420 acgaggtgca ctcgagcttc atccaccggcgcctccgcag ccaggagcgg cgggagatgc 3480 agcgcgagat cctctccatt ttgggcttgccccaccgccc gcgcccgcac ctccagggca 3540 agcacaactc ggcacccatg ttcatgctggacctgtacaa cgccatggcg gtggaggagg 3600 gcggcgggcc cggcggccag ggcttctcctacccctacaa ggccgtcttc agtacccagg 3660 gcccccctct ggccagcctg caagatagccatttcctcac cgacgccgac atggtcatga 3720 gcttcgtcaa cctcggtgag taagggcaggcgagggtacg ccgtctcctt tcgggggcac 3780 tttgagactg ggagggaggg agccgcttcttctatgcagc ccgcccagct ttccgctcct 3840 ggctgaaatc gcagtgcctg cccgagggtctcccacccac agccctatga ctcccaagct 3900 gtgtgcgccc ccaggtcggg ccgctgggtcggtgagcctg taggggttac tgggaaggag 3960 ggatcctccg aagtcccctc catgttacgccgccggccgc atctctgggg ctggaggcaa 4020 gggcgttcaa agcgcggggc tcggtcatgtgagctgtccc gggccggcgc cggtccgtga 4080 cctggatgta aagggccctt cccggcgaggctgccttgcc gcccttcctg ggcccctctc 4140 agccctgcct ggctctggca tcgcggccgtcgcaccccct taccctccct gtcaagccct 4200 acctgtcccc tcgtggtgcg cccgccttaggctaccggcc gctccgagcc ttggggcccc 4260 tctccgggcg ccgatgcccc attctctcttggctggagct ggggaagaaa cggtgccatt 4320 gctaattttc tttgttttct ttctttgtttatttttttct tttttctttt tttttctttt 4380 cttttctttt cttttttttt ttttttgagacggagtttca ctcttgctcg cccagactgg 4440 agtgcaatgg cgcgatctct gctcaccgcaacctctgcct cccgggttca agcgattctc 4500 gtgcctcagc ctcccgagta gctgggattacaggcatgcg caccatgcct ggctaatttt 4560 gtattttagt agagacaggg tttctccatgttaggcaggc tggtctcgaa ctcccgatct 4620 caggtgatcc tcccgcctca gcctcccaaagtggtgctgg gattacaggc gtgaagctgt 4680 gccctgccgc tagtcttcta ttttaagtatttagtggtag gtcccgggcc ggcagaatct 4740 attttcagca tttaccacgt gtggcgcgcaaaccacaggt tttggcgatt gggttgcgcg 4800 ggatctcaga gctgacgacc gcgggggcctgggggtcccg gtttccgact ggagccgcga 4860 cgaccccggc gacggcagcc tggggctgcagccgagggcc ggggagctcc ccctccatat 4920 gtgcgcgcac attctccaga cttgctcaaactaacccccc ggagcagcgc acgggctggg 4980 actgatgatc aaatatttgg tttccgagataacacacccc gatagcgctg tttcctgagc 5040 cgctttcatt ctacttgtgt aacttgctgcgaaaacccga accaagtcaa gacagcaaac 5100 tcacgcccac gggcctgtgt caacatggaaataatgatac tgaagcccca cgctgggcac 5160 ctggggcgtg gactgggggc gcgggggaagcgcagatccg ccttcatgct tcccctcctc 5220 ctgataaggt ccctggagtt cccgggagccattgtctgta cttaataata actaaatcca 5280 actagtgaac caagcttcag cgaggcaaggggagggaggt ttagatgcca aaattacctt 5340 caaaaaagtt taaattatac taagcagccagttaagaagg aagcagcaat atatgacctg 5400 atttagaacc atctccaaga tgtatgaggtggaaagaagc aaggtgcaga tgagtgggct 5460 gcatgtgtgc ttgtatatca tcgtgtcctcctggaggaag acaccaggaa ctggagagag 5520 attttactgg aggggtatat ggcgggggcatagctggggc ttacggagtg ggaggtgggg 5580 tctgattttt cgtcgtctgc acttctgtatttgtgatttt tttaaaacaa tgtgtattta 5640 ttaactatac caaaaaataa aggaaaattccaaatacata catataaata atgaaccgca 5700 gagctctgtc gccctcctga agcctggggttagccagggc cctttctctg gtgggggatt 5760 tatagcatct tcccttctgt tgggtaccccggactcccac tgaatgtgca ggtcccagtg 5820 gctgccttca gagcctggct ggaatcattaaaaaggtatt tgtaatctct ggcttctgca 5880 gaaggccctg caaaccaaga gcaaaaaagcccccagtgct tatgggccgg cagtgtgggc 5940 taggcccggg gctccctgtc cccaagagaaagaccaggtt gctcggaggg tgcctctggg 6000 aactttggtg cgggctattt gctccccccatggcggcagg agcaagctgg gacttgtttg 6060 ggaaggccac agctgggtgg ttttcctcctctggctgtac atacaccttt caatccattt 6120 ctttcatctt gaaaggacaa agaccggcttgtctgagcct cttaatcagt caggctggct 6180 ttgggctttg gggaccctga ctttctcaggtctagctttc tgggacatca ctccaaatta 6240 gatggcagag tggcttttaa cagagcgcactgaccttgtt ttctttctct ctctgtccct 6300 aaactcgagg tcattagtta ggtgaagacctgggctgcag tttggcgaga cacttcctgt 6360 agatgcttct aatgttggcc tttaatttctgctaagcagc agcacacaaa taaatggcct 6420 gtcccttcta tcctgttgta gcttggaatttctccatagg agggacttgg gggtggcagt 6480 agggttggag agggttgggg ggaggtgtaggagacttgtc tggccactga gtttgctgag 6540 aaagtactgc tatagtgttt ttccttggattgcaaatcat gttgatctga actgctgatt 6600 tgaagtggat tgagaggatg gaacaatagaaggaggatat ggctcaggac agtcaagtac 6660 tggaagaggg aaaggtacaa agaggtgttggcactgaatg accctgaaca gggctgccct 6720 ggaaatatca gaggtgagtg acaaagagaactctagtcga aggtctggaa gtcaattatt 6780 gtctccagct tttgtcccac cctaagggatggagcatgaa cttcatgcat gtaacatccc 6840 tccaggagcg ctgaggttct gggaattcccagtgctggct accatgccat tcttttctca 6900 ttcactcaag agcgtattgg gatatgcgtgcatgaaagca atgtaattat gggcacaacc 6960 tcaaaacctg ctctaatttt ttttttttttggagatggag tctcgctcca tcacccaggc 7020 tggagtgcaa tggcgcgatc tcagctcactgcaagctcag acctccaggg ttcacaccat 7080 tctcctgcct cagcctcccg agtagctgggaatacaggcg cccgcaccat gcgcggctaa 7140 tttttttgta tttttagtag agacggggtttcactgtgtt agccaggatg gtctcgatct 7200 cctgacctcg tgatccaccc gcctcggcctcccaaagttc tgggattaca ggcgtgacag 7260 ccgtgcccgg aatctgctct aattttttaaagatatcatt tgcaaacttt gggcacttga 7320 gtcactcagt aagatattat ttacaaccccaccatagatt caaacctctg tcctagaatg 7380 ttgtcgagtt aggcatctgg cttgcagcaacagctggctt tcctgtctat gctgtctcct 7440 tccagggagg atgtttcacc cttcatattgaggaaatggg cacagagaac ccatttctct 7500 tactcatcat gtaacttcag tgggatggtcagatctatct ttaacctggc cactcttcca 7560 caagctcaca ctgactccag caagatcttaaactagaagg caggagttca aatcctagct 7620 ggtgcagtgg ccaaatctcg gctcacagcaccttctgcct cctgggctca agcgatcctc 7680 tgacctcagt ctcccaagta gctgggaccataggcatgca ccactatgcc tggctaattt 7740 ttgtattttt gtaatttttt gtagagacagagtttcacca tgttgcccag cccagtcttg 7800 aactcctgga ctcaagcaat cttcccacctttgcctacca gagtgccggg attacaggtg 7860 tgagccatca tgctagttgc gcacagttgggcgaaactga cagatgagaa agcagaacct 7920 cgtgagtcca ctcagtaaga gactccctactttctttctg agtctttgtt tctcatcaat 7980 tgaatggcaa taaacaactt ggtggcccaagagttgatga caacagtcct ataagattat 8040 acatgtaaaa gaaacagagt attctacaaatatcagttat tgatagttca ataggcaacc 8100 tgacattacc ttttcttgga acttgatgaacaactcagaa actcattaat atcaaaccca 8160 atggtgagca cttggtcttt atttatggctgtaagagaag aaattgaatt aactctatgt 8220 aaatgccaac taagaacatc gaagtctgaaatcaacagtt ttcctcgctc atacgacaca 8280 cccaaactca agcagtggtt ccaagcccctttggaaaata ccatgggcta acgactttaa 8340 aagcttagaa gtgaattcta cttacttattacttaaaagt ggttctcaaa cttcaaggtg 8400 aatcaaaatc atctgtagag cttgttaaaacacaggttgc tggtccaccc caagagtgtc 8460 ttgagtcagt aggtctcaag tagggctcaagaatatgcat ttctaatgag ctccaggtga 8520 gtctaagtgt tagtcgtcgg tcttgggaccacaactttgg gaacaattga tttagaagaa 8580 ctcaaagatc agaaaggggt ggaatatttttaaaattgtg gtaaaatacg cataaacaga 8640 aaaggtacaa ttttaaccac ttagagagaggtgggatcta agaacagaaa ttgttatgcc 8700 atcaaaggtg agttcagata agcattattaaatggtatct atggataaac ttcaggggcc 8760 ctgtggagca acccaatgct gggatggggtccaggtgtgc tatggtttgg atgtggtttg 8820 tccctacaaa aactcatgtt gaaatttaattgccagtgta acattattga gaggttatgg 8880 acttttaaga ggcatttggg tcatgagggatccaccttca gggattagtg cagtctccag 8940 ggagtgagtg agttcccatt ctagtgggactggattagtt accatacagt ggttgttata 9000 aagtgaggct gcttctggtg ttttatctgtttgcaggcac ttccttcccc ttccacttct 9060 ctgccaggtt aggatgcagc atgaggccctcaccagaagc tgaccagatg tggctgcctg 9120 atcttgaact tcccagtccc cagaaccatgagctaaataa accttttttc tctataaatt 9180 acgcagtcta gagtattcta ttatagcaacacaagacaga ctaagacaca gtggtagaaa 9240 gaacactact gacttctccc atactctggcctatggacaa gagtgacaga cagacaagag 9300 tgaatatcag ggccctcagg cacattcctctctgcccctt cctcccttct tgcagagtct 9360 ccagtgactg ccagctaatg ctatcatagaccccaccttt cccctgactt gattggacca 9420 gaagcagcct cctgatccat ggccaacaatcagattcact ttcaagaatt tgaactaaga 9480 gacactagga agatggccct tgagctgtgagtcctacact tgaaagttct tagcatcttg 9540 gtcaggtacc caccagggcc atgtgcaaactgagataatg gggacatgga acaagggtaa 9600 gtggagaggg ctggctggag agagacgggcagaggaaagc cctgccaaga ggagcagaga 9660 tgagagacct tggagggaga ggtaataaaaggaggcaaag atgattttcc atgcttacaa 9720 ctcacagctg aggcctaact atctttatgtccataagagg catccttgtg tcgaacctct 9780 cctctttctt gggtcaatgg gggatggttgcaagggacca tcagtaggaa ggcatagtac 9840 actaacccag tctggggtgg gcttttagactagtcttcct cccatgctcc tcctcccatt 9900 ggaaccccgg actttcaaga ctgctacctagcacaccagt gcaccagatg tcactcaaaa 9960 cctcttcagc aatggcccac tcaccttcaaaaaggctgaa gagcagactg gctgggttct 10020 tcatggtgga ggggcagtct gggaggttttaaggttgaag atgaaaactt tcacttttgg 10080 ctcaatggtc tgaaaaagag aaggaccagcaagtgaactg aagcctcctg gaaagcatct 10140 tgataacagg ggcagagttt caagatgagaagctgtggca cttactctgg ctttggaaat 10200 gacctctaag tatctcagtt aattaaaggagtcaaactct agactcgaag gagaagatct 10260 acaattttca ataacatagt ctaccctcccctccttcccc caccttcacc tcttctttca 10320 tcacaggctt acagggcacc tcttagagccaggcacggtg ttgggatcag gaacaaggcc 10380 actgctcaca tccagagcct gtgctacttaagaagcttcc aggacctctt ggatggctgt 10440 ggttagtgcc ctacttttcc cagcaggttggatgcagaat catgctcttg tcgttcagga 10500 tgaccatggg gaccatgggt ctgagcctgtgaccctccag tctacagtgt gttggtgagg 10560 aaggagcagt tgtcactggg gtcactggcaatgggcatgc ctccatctag cttaggcaag 10620 atgcttagac tcagagccag agagtgaaacccagacacta atgagctgtc ggtgttggtg 10680 tgtgttctct tcctcttcca gtggaacatgacaaggaatt cttccaccca cgctaccacc 10740 atcgagagtt ccggtttgat ctttccaagatcccagaagg ggaagctgtc acggcagccg 10800 aattccggat ctacaaggac tacatccgggaacgcttcga caatgagacg ttccggatca 10860 gcgtttatca ggtgctccag gagcacttgggcaggtgggt gctatacggg tatctgggag 10920 aggtgctgag tttcctctgg gggcagaggaagaaggtggt gagggtttcc ctcccctccc 10980 accccatgag ctctgcttcc catctgttggggtagtggag ctgtgacctg ctaacgcgaa 11040 gcccgtgtct ctcctcctct ctcgcagggaatcggatctc ttcctgctcg acagccgtac 11100 ctctgggcct cggaggaggg ctggctggtgtttgacatca cagccaccag caaccactgg 11160 gtggtcaatc cgcggcacaa cctgggcctgcagctctcgg tggagacgct ggatggtgag 11220 tcccccgcca ctgccagtcc taatgcagcctgtgctcctg gacttcagga gggtctcagc 11280 agtgctcatg cttgcttcac tacaaacaggcttccccgcc cctcccaacc agtactccat 11340 gttcagcctt ttgatcctgc agccctgtcccgctcgtggc cctcctgtaa ctgctcttct 11400 gtgcacttgg ctgcttcctg tccagggcagacgatcaacc ccaagttggc gggcctgatt 11460 gggcggcacg ggccccagaa caagcagcccttcatggtgg ctttcttcaa ggccacggag 11520 gtccacttcc gcagcatccg gtccacggggagcaaacagc gcagccagaa ccgctccaag 11580 acgcccaaga accaggaagc cctcggatggccaacgtggc agggtatctt aggtgggagg 11640 gatcacagac ccaccacagg aacccagcaggccccggcga ccgcaggaga ctgactaaaa 11700 tcattcagtg ctcaccaaga tgctctgagctctcttcgat tttagcaaac caggagtccg 11760 aagatctaag gagagctggg ggtttgactccgagagctcg agcagtcccc aagacctggt 11820 cttgactcac gagttagact ccactcagaggctgactgtc tccagggtct acacctctaa 11880 gggcgacact gggctcaagc agactgccgttttctatatg ggatgagcct tcacagggca 11940 gccagttggg atgggttgag gtttggctgtagacatcaga aacccaagtc aaatgcgctt 12000 caaccagtag aaaattcacc agcccgcagagctaaggttg ggtggacatt agggttggtt 12060 gatccaggag ctcaacagtg tcctctgagccccagctcct tctgccccac cccaccatct 12120 tcagtgctgc ttcctctcaa ggccacagctgtagttggcc aggggggctt cattattttt 12180 tgctcctggg cagtaggagg aagagaatgaatgtctctcc atgggtcttt cttaggaatg 12240 tgggaacttt ttccagaagt ctctatgtcttttagtttgt gttgggtcac ttgcccttcc 12300 tgaaccactt cctgactcct ggacaggatgtgcactgatg agcttagctt tggggatcta 12360 atagtgactt tacaaagcct ctttgagaaggtgacattgg aaccaaggct tgagcagaca 12420 caacaaagat tgcagggagg ggcattgcaggtggaggaaa cggcacatgc aagagccctg 12480 cgtgggagtg agcttggtgt ttggtcaatcagttgtcaga gcacaccggg ccctgtcagc 12540 aggcacagcc tgggcctgct ctgagtatgacagagagccc ctgggaagtt gtaggtggag 12600 gaaagacagg tcatgactag gaaaaaagcaatccctctgt tgtggggtgg aaggaaggtt 12660 gcagtgtgtg tgagagagag acaagacagacagacagaca cttctcaatg tttacaagtg 12720 ctcaggccct gacccgaatg cttccaaatttacgtagttc tggaaaaccc cctgtatcat 12780 tttcactact caaagaaacc tcgggagtgttttcttctga aaggtcatca ggttttgact 12840 ctctgctgtc tcatttcttc ttgctggtggtggtgatggt tgcttgtccc aggccctgtc 12900 ccgcatcctc ttgcccctgc agagggatgagtgtgttggg gcctcacgag ttgaggttgt 12960 tcataagcag atctctttga gcagggcgcctgcagtggcc ttgtgtgagg ctggaggggt 13020 ttcgattccc ttatggaatc caggcagatgtagcatttaa acaacacacg tgtataaaag 13080 aaaccagtgt ccgcagaagg ttccagaaagtattatggga taagactaca tgagagagga 13140 atggggcatt ggcacctccc ttagtagggcctttgctggg ggtagaaatg agttttaagg 13200 caggttagac cctcgaactg gcttttgaatcgggaaattt accccccagc cgttctgtgc 13260 ttcattgctg ttcacatcac tgcctaagatggaggaactt tgatgtgtgt gtgtttcttt 13320 ctcctcactg ggctctgctt cttcacttccttgtcaatgc agagaacagc agcaggcacc 13380 agaggcaggc cttgtaagaa gcacgagctgtatgtcagct tccgagacct gggctggcag 13440 gtaaggggct ggctgggtct gtcttgggtgtgggccctct ggcgtgggct cccacaggca 13500 gcgggtgctg tgctcagtct tgtttctcatctctgccagt taagactcca gtatcaagtg 13560 gcctcgctag ggaaggggac ttgggctaaggatacaggga ggcctcatga aatccgagag 13620 cagaaatgtg gttgagactt gaactcgaaccaggaaccca aacactttgg actctgaacc 13680 ccattctctg catgcacctc attcccatcccttggctggc tgcttctcaa gatgatgccg 13740 ggccgtgtgt ttgaatgtag atacctggggagccatctcc ccctctgccc tctgacttca 13800 tttaccccat tcccattccc acgggagggacggatctccc cagcttggtt caggcgcttg 13860 ttcctgaacc agtcaactgt ttcaggggtggggtcatgtt actggcacat ggctgccccc 13920 tctggagcca tttgcatgga gtgaggcaaaaggcagggga tgaatctagg agaggagtga 13980 gggtcatgtg atccacctgc cgtgagctctggatcgtgat tctcattcag cagtcacgag 14040 catctcgagc gttctgggcc ctgttctaggtactggattg gagatgcagc gatgaacact 14100 gcaatgtgtc tgccctgtgg ggctcaaatatccctggaga gggtattgtc atgaggtcat 14160 cagggcaact ggtggtattc taccctcagggagcttgtag ttcagtggga gagtccagaa 14220 tcttccctgg ggattatgcc cagacacactcagggcgtac gtgcacacag ccagctctga 14280 gccctcctgt gagcctgccc tcaggactgatgaccacatc tacctgcagc tgggacagaa 14340 cccaaactcc aggggcctct gctggaagattccatgtgct taagcatcac tgaggagtat 14400 attgattatt gggcaacatt tctgtgccacccagacccta gaggcaagga tggcacatgg 14460 atcccttact gaccagtgca cccggagccagcatgggtga tgccattatg agttattagc 14520 ctctctggca ggtgggcaaa ccgaggcatggaggtttgtt taaggtgaac tgccagtgtg 14580 tgaccaccta gtgggggtag agctgatgattgcctcacac cggaggctcc ttcctgtgcc 14640 gcgttctgtc cagaagacac agccatggatgtccatttta ggatcagcca agcccgtggg 14700 gctttccttc atttttattt tatgtttttttagaaatggg gtcttgctct gtcacccagg 14760 ctggggtgca gtggtgtgat catacgtcaccgcagctttg agccgtcttc ccactcagtc 14820 tactaagctt ggactatagg ccaagactatagagtggtcc ttctttccat tcttttggga 14880 ccatgagagg ccacccatgt ttcctgcccctgctgggccc tgctgctcag aaggcatggt 14940 ctgaggcttt caccttggtc gtgagccttcgtggtggttt ctttcagcat ggggttggga 15000 tgctgtgctc aggcttctgc atggtttcccacactctctt ctcctcctca ggactggatc 15060 atcgcgcctg aaggctacgc gcgctactactgtgaggggg agtgtgcctt ccctctgaac 15120 tcctacatga acgccaccaa ccacgccatcgtgcagacgc tggtgggtgt cacgccatct 15180 tggggtgtgg tcacctgggc cgggcaggctgcggggccac cagatcctgc tgcctccaag 15240 ctggggcctg agtagatgtc agcccattgccatgtcatga cttttggggg ccccttgcgc 15300 cgttaaaaaa aaatcaaaaa ttgtactttatgactggttt ggtataaaga ggagtataat 15360 cttcgaccct ggagttcatt tatttctcctaatttttaaa gtaactaaaa gttgtatggg 15420 ctcctttgag gatgcttgta gtattgtgggtgctggttac ggtgcctaag agcactgggc 15480 ccctgcttca ttttccagta gaggaaacaggtaaacagat gagaaatttc agtgaggggc 15540 acagtgatca gaagcgggcc agcaggataatgggatggag agatgagtgg ggacccatgg 15600 gccatttcaa gttaaatttc agtcgggtcaccaggaagat tccatgtgat aatgagatta 15660 acgtgcccag tcacggcgac actcagtaggtgttattcct gctctgccaa cagcaaccat 15720 agttgataag agctgttagg gattttgtccttttgcttag aatccaaggt tcaaggacct 15780 tggttatgta gctccctgtc atgaacatcatctgagcctt tcctgcctac tgatcatcca 15840 ccctgccttg aatgcttcta gtgacagagagctcactacc aggactactc cctcctttca 15900 tttagtaatc tgcctccttc ttttcttgtccctgtcctgt gtgttaagtc ctggagaaaa 15960 atctcatcta tccctttcat ttgattctgctctttgaggg caggggtttt tgtttctttg 16020 tttgtttttt taagtgttgg ttttccaaagcccttgctcc cctcctcaat tgaaacttca 16080 aagccctcat tgggattgaa ggtccttaggctggaaacag aagagtcctc cccaacctgt 16140 tccctggcct ggatgtgctg tgctgtgccagtatcccctg gaaggtgcca ggcatgtctc 16200 cccggctgcc aggggacaca tctctatccttctccaaccc ctgccttcat ggcccatgga 16260 acaggagtgc catcgccctg tgtgcacctacttccatcag tatttcacca gagatctgca 16320 ggatcaaagt gaattctcca gggattgtgaaatgatgcga ttgtggtcat gtttaaaagg 16380 gggcaactgt cttctagaga gtcctgatgaaatgcttcca gaggaaatga gctgatggct 16440 ggaatttgct ttaaaatcat tcaaggtggagcaggtgggg aagggtatgg atgtgtaaga 16500 gtttgaaatt gtccatcata aaatgtgtaaaaagcatgct ggcctatgtc agcagtcaca 16560 gcctggaggt ggtaacagag tgccagtcactgatgctcaa gcctggcacc tacagttgct 16620 ggaaacccag aagtttcacg ttgaaaacaacaggacagtg gaatctctgg ccctgtcttg 16680 aacacgtggc agatctgcta acactgatcttggttggctg ccgtcagctt aggttgagtg 16740 gcggtcttcc cttagtttgc ttagtccccgctattcccta ttgtcttacc tcggtctatt 16800 ttgcttatca gtggacctca cgaggcactcataggcattt gagtctatgt gtccctgtcc 16860 cacatcctct gtaaggtgca gagaagtccatgagcaagat ggagcacttc tagtgggtcc 16920 aagtcaggga cactattcag caatctacagtgcacagggc agttccccaa cagagaatta 16980 cctggtcctg aatgtcggat ctggccccttccttccccac tgtataatgt gaaaacctct 17040 atgctttgtt ccccttgtct gcaaaacagggataatccca gaactgagtt gtccatgtaa 17100 agtgcttaga acagggagtg cttggcttggggagtgtcac ctgcagtcat tcattatgcc 17160 cagacaggat gtttctttat agaaacgtggaggccagtta gaacgactca ccgcttctca 17220 ccactgccca tgttttggtg tgtgtttcaggtccacttca tcaacccgga aacggtgccc 17280 aagccctgct gtgcgcccac gcagctcaatgccatctccg tcctctactt cgatgacagc 17340 tccaacgtca tcctgaagaa atacagaaacatggtggtcc gggcctgtgg ctgccactag 17400 ctcctccgag aattc 17415 2 17 DNAArtificial Sequence Description of Artificial Sequence ConsensusSequence 2 ttcacgcwts antkmny 17 3 11 DNA Artificial SequenceDescription of Artificial Sequence Consensus Sequence 3 gtcaygcrtg a 114 102 PRT Artificial Sequence Description of Artificial Sequence Genericsequence OPX 4 Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu GlyTrp Xaa 1 5 10 15 Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr TyrCys Glu Gly 20 25 30 Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala ThrAsn His Ala 35 40 45 Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa XaaVal Pro Lys 50 55 60 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser ValLeu Tyr Xaa 65 70 75 80 Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys Xaa ArgAsn Met Val Val 85 90 95 Xaa Ala Cys Gly Cys His 100 5 97 PRT ArtificialSequence Description of Artificial Sequence Generic morphogenic sequence5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 1015 Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa 20 2530 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 4045 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro 50 5560 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 7075 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 8590 95 Xaa 6 102 PRT Artificial Sequence Description of ArtificialSequence Generic morphogenic sequence 6 Cys Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Pro XaaXaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Cys Xaa Pro Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Cys Xaa Cys Xaa100 7 5 PRT Artificial Sequence Description of Artificial SequenceGeneric morphogenic sequence 7 Cys Xaa Xaa Xaa Xaa 1 5 8 16 DNAArtificial Sequence Pax 6 consensus sequence 8 annttcacgc atgant 16 9 11DNA Artificial Sequence Pax 6 consensus sequence 9 ttcacgcatg a 11 10 30DNA Homo sapiens 10 aagcttgatg cctgcacagt cagccctcag 30 11 30 DNAArtificial Sequence Reverse compliment primer 11 catcgcgccg gatccacgcgctacccgggc 30 12 30 DNA Homo sapiens 12 gcccgggtag cgcgtagagc cggcgcgatg30 13 30 DNA Artificial Sequence Mutagenesis altered OP-1 sequence 13gcccgggtag cgcgtggatc cggcgcgatg 30 14 57 DNA Artificial SequenceAltered OP-1 promoter in construct pAS3.3 14 gcccgggtag cgcgtggatctaagtaagct tggcattccg gtactgttgg taaaatg 57 15 10 DNA ArtificialSequence nucleic acid 15 tcacgcntga 10

What is claimed is:
 1. A method for identifying a candidate compound forthe ability to modulate expression of OP-1, said method comprising thesteps of: (a) incubating a candidate compound with a cell co-transfectedwith vectors comprising: (i) a DNA sequence encoding a reporter gene inoperative association with at least one OP-1-specific non-codingsequence which can modulate expression of said reporter gene, whereinsaid non-coding sequence comprises a first Pax-responsive OP-1modulating element which is responsive to a first Pax gene expressionproduct; and (ii) a DNA sequence encoding a first Pax gene expressionproduct; (b) measuring the level of reporter gene expressed in saidcell; and (c) comparing said level with that of said reporter geneexpressed in said cell in the absence of said candidate compound,wherein a change in reporter gene expression level is indicative of saidcandidate's ability to modulate OP-1 expression.
 2. The method of claim1 wherein in step (a)(i) the DNA sequence encoding the reporter gene isfurther in association with a second Pax responsive OP-1 modulatingelement, and wherein in step (a) the cell has also been transfected witha DNA sequence encoding a second Pax gene expression product.
 3. Themethod of claim 2 wherein said first Pax responsive OP-1 modulatingelement is selected from the group of DNA sequences corresponding tobases 108-121, 139-154, 157-167, 365-378, 497-511, 598-613, 1123-1140,1144-1161, 1285-1297, 1750-1762, 2001-2023, 2365-2378, or 2931-2944 ofSEQ. ID No. 1, and wherein said second Pax responsive OP-1 modulatingelement is selected from the group of DNA sequences corresponding tobases 491-503, 737-747, 891-903, or 994-1006 of SEQ. ID. No.
 1. 4. Themethod of claim 2, wherein said first Pax responsive modulating elementis a Pax 6 responsive modulating element and said second Pax responsivemodulating element is a Pax 2 responsive modulating element, and whereinsaid first Pax gene expression product is Pax 6 and said second Paxexpression product is Pax
 2. 5. The method of claim 1, wherein saidfirst Pax-responsive OP-1 modulating element is a Pax 6-responsivemodulating element, and wherein said first Pax gene expression productis Pax
 6. 6. The method of claim 5, wherein said Pax 6 responsivemodulating element comprises a sequence corresponding to one of thefollowing series of bases of SEQ ID NO:1: 108-121, 139-154, 157-167,365-378, 497-511, 598-613, 1123-1140, 1144-1161, 1285-1297, 1750-1762,2001-2023, 2365-2378, or 2931-2944.
 7. The method of claim 5, whereinsaid Pax 6 responsive modulating element comprises SEQ ID NO:2, SEQ IDNO:8 or SEQ ID NO:9.
 8. The method of claim 1, wherein said firstPax-responsive OP-1 modulating element is a Pax 2-responsive modulatingelement, and wherein said first Pax gene expression product is Pax
 2. 9.The method of claim 8, wherein said Pax 2-responsive modulating elementcomprises a sequence corresponding to one of the following series ofbases of SEQ ID NO:1: 491-503, 737-747, 891-903, or 994-1006.
 10. Themethod of claim 8, wherein said Pax 2 responsive OP-1 modulating elementcomprises SEQ ID NO:3, or SEQ ID NO:15.
 11. The method of claim 1,wherein said first Pax-responsive OP-1 modulating element comprises asequence corresponding to one of the following series of bases of SEQ IDNO:1: 1-2073, 1-1297, 1-2691, 1-378, 491-1006, 1750-2023, 1750-2378,1750-2691, or 1750-2944.
 12. An isolated cell transfected with (a) a DNAencoding a reporter gene in operative association with at least one OP-1specific non-coding sequence, wherein said OP-1 specific non-codingsequence comprises a first Pax-responsive modulating element which isresponsive to a first Pax gene expression product and (b) a DNA encodinga first Pax gene expression product.